3'-end labeling of RNA with recombinant yeast poly(A) polymerase.

Two commonly used methods to end-label RNA-molecules are 5'-end labeling by polynucleotide kinase and 3'-end labeling with pCp and T4 RNA ligase. We show here that RNA 3'-ends can also be labeled with the chain-terminating analogue cordycepin 5'-triphosphate (3'-deoxy-ATP) which is added by poly(A) polymerase. For a synthetic RNA it is shown that 40% of cordycepin becomes incorporated when the nucleotide is used at limiting concentrations and that with an excess of cordycepin 5'-triphosphate essentially all the RNA becomes modified at its 3'-end. The reaction is complete within minutes and the RNA product is uniform and suitable for sequence analysis. The efficiency of labeling varies with different RNA-molecules and is different from RNA ligase. Poly(A) polymerase preferentially labels longer RNA-molecules whereas short RNA-molecules are labeled more efficiently by T4 RNA ligase.     

Primer RNA for DNA synthesis on single-stranded DNA template in a cell free system from Drosophila melanogaster embryos

A cytoplasmic extract of Drosophila melanogaster early embryos supported DNA synthesis which was dependent on an added single stranded DNA template, phi X174 viral DNA. The product DNA made during early reaction was about 100 to 600 nucleotides in length and complementary to the added template. After alkali treatment, 70 to 80 per cent of the product DNA chains exposed 5'-hydroxyl ends, suggesting covalent linkage of primer RNA at their 5'-ends. Post-labeling of 5'-ends of the product DNA with polynucleotide kinase and [gamma-32P]ATP revealed that oligoribonucleotides, mainly hexa- and heptanucleotides, were covalently linked to the 5'-ends of the majority of the DNA chains. The nucleotide sequence of the linked RNA was mainly 5'(p)ppApA(prN)4-5, where tri- (or di-) phosphate terminus was detected by the acceptor activity for the cap structure with guanylyltransferase and [alpha-32P]GTP. The structure of this primer RNA was comparable to that of the octaribonucleotide primer isolated from the nuclei of Drosophila early embryos.     

A 32P postlabeling assay for determining the incorporation of bromodeoxyuridine into cellular DNA

Randerath's procedure for 32P postlabeling of 3'-monophosphate deoxyribonucleotides from digests of cellular DNA has been modified. 3'-Monophosphate deoxyribonucleotides are converted to 3',5'-bis[32P]phosphate deoxyribonucleotides with polynucleotide kinase and [32P]ATP; these products are enzymatically converted by P1 nuclease and polynucleotide kinase into 5'-[32P]monophosphate deoxyribonucleotides, which are separated from [32P]ATP on an anion-exchange column eluted with 0.1 M NaH2PO4, pH 6.5. Labeled mononucleotides in the effluent are separated by high-performance liquid chromatography. Values for the base composition of calf thymus DNA determined with this modified assay compare very favorably with reported values. The assay was used to measure the level of incorporation of the clinically useful agent bromodeoxyuridine into the DNA of 9L rat brain tumor cells. The modified assay appears to be a very accurate method for the determination of levels of base analogs incorporated into DNA.     

Isolating and sequencing the infrequent 3'-ends of a specific mRNA

Procedures are described for identification of very infrequent in vivo 3'-ends of RNA. After purification by filter hybridization, the 3'-ends were labeled with [5'-32P] cytosine-3'-P in the RNA ligase reaction. Significantly fewer counts were incorporated in the ligase reaction than in the polynucleotide kinase reaction to label 5'-ends. The incorporation was increased by increasing the RNA concentration 5-10 fold by using only one round of filter hybridization. Non-specific RNA binding could be eliminated by RNase A treatment of the filter if a great excess of denatured heterologous DNA was immobilized along with the DNA probe. Significant amounts of DNA were released when eluting the hybrid RNA from such filters. DNA inhibited the ligase reaction, while its DNase products were even more inhibitory. Treatment of the DNase products with alkaline phosphatase completely eliminated the inhibition. We detected no spurious 5'- or 3'-ends generated in the hybrid RNA by RNase A activity used to reduce the non-specific RNA. Also, RNase T1 could be used in place of RNase A to eliminate non-specific RNA binding, but about 25 times more RNase T1 (microgram/microgram) was needed. We used partial alkali digestion to sequence 3'-ends. A major (one hit) and minor (two hit) set of products were produced which could be distinguished from each other by alkaline phosphatase treatment and homochromatography of the products.     

A new and improved method for 3′-end labelling DNA using [α-32P]ddATP

We have synthesised dideoxyadenosine-5′-[α-³²P]triphosphate ([α-³²P]ddATP) at a specific activity of 3000 Ci/mmol and directly compared it with cordycepin-5′-[α-³²P]triphosphate ([α-³²P]KTP) as a means to 3′-end label DNA. The [α-³²P]ddATP was found to be three to five times more efficient than [α-³²P]KTP. Blunt and 3′-protruding ends were labelled more efficiently with [α-³²P]ddATP using terminal transferase than were the 5′-ends with [γ-³²P]ATP using polynucleotide kinase by standard methods. This improvement in efficiency of labelling DNA and the simplicity of the method allows 3′-end labelling of DNA to become a realistic alternative to 5′-end labelling. We have also compared [α-³²P]ddATP- and [α-³²P]KTP-labelled DNA in Maxam and Gilbert sequencing procedures and find that both give equally good results.     

Rapid decreases in phosphatidylinositol in isolated luteal plasma membranes after stimulation by luteinizing hormone

Phospholipid concentrations were determined in plasma membrane preparations from porcine corpora lutea after incubation for 15 to 120 s without or with 0.5 microgram/ml luteinizing hormone (LH) or 2 microM dibutyryl cyclic adenosine 3',5'-monophosphate (dbcAMP). Treatment with LH caused a dramatic loss of 9 nmol in plasma membrane phosphatidylinositol (PI)/mg protein after 15 s of incubation, but no significant changes in other measurable phospholipids. Also, phospholipid concentrations were unchanged in untreated and dbcAMP-treated plasma membranes. The nature of the LH-induced decrease in PI was studied by incubating plasma membrane preparations for 15 s with [gamma 32P] adenosine 3',5'-triphosphate (ATP). 32P was incorporated only into three phospholipids: phosphatidic acid, phosphatidylinositol 4'-phosphate (PIP), and phosphatidylinositol 4',5'-bisphosphate (PIP2). Although LH generated small but significant increases in labeling of PIP and PIP2, less than 0.5 nmol of total phospholipids/mg protein were radiolabeled in 15 s. Phosphatidylinositol kinase activity, the enzyme that converts PI into PIP, was not affected by LH or dbcAMP treatment. However, incubation of luteal plasma membranes for 15 s with LH resulted in an increase of approximately 2 nmol 1,2-diacylglycerol/mg protein more than that observed in untreated or dbcAMP-treated plasma membranes. In summary, these experiments suggest that LH may stimulate hydrolysis of PI (and possibly PIP and PIP2) in isolated luteal plasma membranes.     

2- and 8-azido photoaffinity probes. 1. Enzymatic synthesis, characterization, and biological properties of 2- and 8-azido photoprobes of 2-5A and photolabeling of 2-5A binding proteins

The 2- and 8-azido trimer 5'-triphosphate photoprobes of 2-5A have been enzymatically synthesized from [gamma-32P]2-azidoATP and [alpha-32P]8-azidoATP by 2-5A synthetase from rabbit reticulocyte lysates. Identification and structural determination of the 2- and 8-azido adenylate trimer 5'-triphosphates were accomplished by enzymatic hydrolyses with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase. Hydrolysis products were identified by HPLC and PEI-cellulose TLC analyses. The 8-azido photoprobe of 2-5A displaces p3A4[32P]pCp from RNase L with affinity equivalent to p3A3 (IC50 = 2 X 10(-9) M in radiobinding assays). The 8-azido photoprobe also activates RNase L to hydrolyze poly(U) [32P]pCp 50% at 7 X 10(-9) M in core-cellulose assays. The 2- and 8-azido photoprobes and authentic p3A3 activate RNase L to cleave 28S and 18S rRNA to specific cleavage products at 10(-9) M in rRNA cleavage assays. The nucleotide binding site(s) of RNase L and/or other 2-5A binding proteins in extracts of interferon-treated L929 cells were investigated by photoaffinity labeling. Dramatically different photolabeling patterns were observed with the 2- and 8-azido photoprobes. The [gamma-32P]2-azido adenylate trimer 5'-triphosphate photolabels only one polypeptide with a molecular weight of 185,000 as determined by SDS gel electrophoresis, whereas the [alpha-32P]8-azido adenylate trimer 5'-triphosphate covalently photolabels six polypeptides with molecular weights of 46,000, 63,000, 80,000, 89,000, 109,000, and 158,000. Evidence that the photolabeling by 2- and 8-azido 2-5A photoprobes was highly specific for the p3A3 allosteric binding site was obtained as follows.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromatin 3''-phosphatase/5''-OH kinase cannot transfer phosphate from 3'' to 5'' across a strand nick in DNA.

Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly (dA) and oligo (dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo (dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'-phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo (dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which cannot be repaired by DNA ligase directly into a 3'-OH/5'-phosphate nick which can be repaired by DNA ligase; the chromatin enzyme rather acts in two steps: hydrolysis of the 3'-phosphate followed by ATP-mediated phosphorylation of the 5'-OH end.     

A binary system of photoreagents for high-efficiency labeling of DNA polymerases

To increase the efficiency of photoaffinity labeling of DNA polymerases, a binary system of photoaffinity reagents was applied. Photoreactive radioactive primers were synthesized by DNA polymerases beta (pol beta) or DNA polymerase from Thermus thermophilus (pol Tte) using a template-primer duplex in the presence of a dTTP analogue containing 4-azidotetrafluorobenzoyl group linked via spacers of varying length to 5-position of uridine ring- 5-[N-(2,3,5,6-tetrafluoro-4-azidobenzoyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-4-dUTP) or 5-[N-[[(2,3,5,6-tetrafluoro-4-azidobenzoyl)-butanoyl]-amino]-trans-3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (FAB-9-dUTP). The reaction mixtures were UV irradiated (lambda = 365-450 nm) in the absence or presence of a dTTP analog, containing a pyrene moiety-5-[N-(4-(1-pyrenyl)-butylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr- 8-dUTP) or 5-[N-(4-(1-pyrenyl)-ethylcarbonyl)-amino-trans-propenyl-1]-2'-deoxyuridine-5'-triphosphate (Pyr-6-dUTP). The most efficient crosslinking of both DNA polymerases was observed in the case of photoreactive DNA primer, carrying the FAB-4-dUMP moiety at the 3'-end, and Pyr-6-dUTP as a sensitizer. The binary system of photoaffinity reagents allows increasing photoaffinity labeling of the both DNA polymerases in comparison to the primer crosslinking without photosensitizer.     

Phosphorylation of Okazaki-like DNA fragments in mammalian cells and role of polyamines in the processing of this DNA.

In mammalian cells DNA synthesis is more complicated than in prokaryotes and less well understood. Here we incubated intact mammalian cells (polyamine auxotrophic Chinese hamster ovary cells and primary human fibroblasts) with [32P]orthophosphate and found that, besides high molecular weight DNA, a species of low molecular weight DNA, approximately 450 bp in size, became efficiently labeled. The short DNA was labeled first, and in pulse-chase experiments the labeling was transient. The isolated small DNA fragments (RNase A-treated) were phosphorylated by T4 polynucleotide kinase specific for polynucleotides with 5'-OH ends. A polynucleotide kinase phosphorylating these DNA pieces was also detected in nuclear extracts of the cells. Treatment with alkaline phosphatase removed most of the 32P label incorporated into the small DNA in vivo. Labeling with deoxyribonucleosides did not reveal these fragments. We hypothesize that the low molecular weight DNA represents Okazaki fragments and that the mammalian DNA replication machinery includes a polynucleotide kinase phosphorylating the 5'-termini of Okazaki fragments. This would imply a novel step in DNA synthesis. We also show that depriving cells of polyamines reversibly blocks synthesis of high molecular weight DNA and leads to accumulation of the short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments.     

Identification, characterization, and quantitative measurement of cyclic AMP receptor proteins in cytosol of various tissues using a photoaffinity ligand.

Two protein bands, present in cytosol fractions from each of seven rat tissues examined, specifically incorporated 32P-labeled 8-azidoadenosine 3':5'-monophosphate (8-N3-[32P]cAMP), a photoaffinity label for cAMP-binding sites. These proteins had apparent molecular weights of 47,000 and 54,000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. These two proteins were characterized in three of the tissues, namely, heart, uterus, and liver, by the total amount of 8-N3-[32P]cAMP incorporation, by the dissociation constant (Kd) for 8-N3-[32P]cAMP, and by the nucleotide specific inhibition of 8-N3-[32P]cAMP incorporation. Several lines of evidence were obtained that the protein with an apparent molecular weight of 47,000 represents the regulatory subunit of a type I cAMP-dependent protein kinase, while the protein with an apparent molecular weight of 54,000 represents the regulatory subunit of a type II cAMP-dependent protein kinase. Almost all of the cAMP receptor protein found in the cytosol of these tissues, as measured by 8-N3-[32P]cAMP incorporation, was associated with these two protein kinases, in agreement with the idea that most effects of cAMP are mediated through protein kinases. The photoaffinity labeling with 8-N3-[32P]cAMP can be used to estimate quantitatively the amounts of regulatory subunit of type I and type II cAMP-dependent protein kinases in various tissues.     

New reagents for affinity modification of biopolymers. Photoaffinity modification of Tte-DNA polymerase]

Arylazides N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-beta-alanine (Ia) and N-(4-azido-2,5-difluoro-3-chloropyridinyl-6)-glycine (Ib) were synthesized and covalently attached to 5-(3-aminopropenyl-1)-dUTP through the amino group to give 5'-triphosphate (IIa) and 5'-triphosphate (IIb). The resulting azides were subjected to photolysis in aqueous solution. The spectral and photochemical characteristics of azides (I) and (II) imply that their use for the modification of biopolymers holds promise. Compounds (IIa, b) effectively substituted dTTP in DNA polymerization catalyzed by thermostable DNA polymerase from Thermus thermophilus B-35 (Tte DNA polymerase). Photoaffinity modification of Tte DNA polymerase was carried out by dTTP analogues (IIa, b) and by earlier obtained 5-[N-(5-azido-2-nitrobenzoyl)-trans-3-aminopropenyl-1]deoxyuridine 5'-triphosphate (III) and 5-[N-(4-azido-2,3,5,6-tetrafluorobenzyol)-trans-3- aminopropenyl-1]deoxyuridine 5'-triphosphate (IV) using two variants of labeling. All four dTTP analogues were shown to modify Tte DNA polymerase.     

A photoreactive analogue of 2'3'-dideoxyuridine 5'-triphosphate: preparation and use for photoaffinity modification of human replication protein A

A new reagent for photoaffinity modification of biopolymers, 5-[E-N-(2-nitro-5-azidobenzoyl)-3-amino-1-propen-1-yl]-2',3'-dideoxyuridine 5'-triphosphate (NAB-ddUTP), was synthesized. Like a similar derivative of 2'-deoxyuridine 5'-triphosphate (NAB-dUTP), it was shown to be able to effectively substitute for dTTP in the synthesis of DNA catalyzed by eukaryotic DNA polymerase beta and to terminate DNA synthesis. A 5'-32P-labeled primer with a photoreactive group at the 3'-terminus was derived from NAB-ddUTP and used for photoaffinity labeling of the human replication protein A (RPA). The covalent attachment of RPA p32 and p70 subunits to the labeled primers was demonstrated. NAB-ddUTP is a promising tool for studying the interaction of proteins of the replicative complex with NA in cellular extracts and living cells during the termination of DNA synthesis.     

RPA subunit arrangement near the 3'-end of the primer is modulated by the length of the template strand and cooperative protein interactions.

To analyze the interaction of human replication protein A (RPA) and its subunits with the DNA template-primer junction in the DNA replication fork, we designed several template-primer systems differing in the size of the single-stranded template tail (4, 9, 13, 14, 19 and 31 nt). Base substituted photoreactive dNTP analogs-5-[ N -(2-nitro-5-azidobenzoyl)- trans -3-amino-propenyl-1]-2'-deoxyuridine-5'-triphosphate (NAB-4-dUTP) and 5-[ N -[ N -(2-nitro-5-azidobenzoyl)glycyl]- trans -3-aminopropenyl-1]-2'-deoxyuridine-5'-triphosphate (NAB-7-dUTP)-were used as substrates for elongation of radiolabeled primer-template by DNA polymerases in the presence or absence of RPA. Subsequent UV crosslinking showed that the pattern of p32 and p70 RPA subunit labeling, and consequently their interaction with the template-primer junction, is strongly dependent on the template extension length at a particular RPA concentration, as well as on the ratio of RPA to template concentration. Our results suggest a model of changes in the RPA configuration modulating by the length of the template extension in the course of nascent DNA synthesis.     

Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate

Photoaffinity labeling of the cAMP receptor protein (CRP) of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has been demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analogue. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5'AMP. The data indicate that (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, one-third remains associated with the amino-proximal alpha core region of CRP [Eilen, E., Pampeno, C., & Krakow, J.S. (1978) Biochemistry 17, 2469] which contains the cAMP binding domain; the remaining two-thirds of the label associated with the beta region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the molecular weight 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. These results suggest that a part of the carboxyl-proximal region is folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.     

Non-canonical nucleotide exchange catalyzed by Escherichia coli DNA-polymerase I and the two-center model of the mechanism of action of this enzyme

It is shown, that DNA hydrolysis catalyzed by E. coli DNA polymerase I is inhibited, when a reaction mixture contains one type of deoxynucleoside 5'-triphosphate (dNTP). When the reaction mixture contains [32P]dNTP, then [32P] is incorporated into DNA and v. v. (32P) from DNA is transferred into dNTP. The nucleotide exchange between DNA and dNTP in the assay mixture is observed only in the case, when the chemical nature of nucleotide residue of dNTP and that of the 3'-terminus of DNA is the same. Analysis of products of DNA hydrolysis in the presence of one type of dNTP using electrophoresis in polyacrylamide gel shows that most of the DNA molecules are terminated at the 3'-termini by the dNMP residue of the same chemical nature as the dNTP in the assay mixture. However, in some cases DNA molecules contain one additional nucleotide residue. This phenomenon can be explained by incorporation of one additional dNMP residue originating from dNTP only in those cases, when a non-typical base pairing of this nucleotide residue with a template residue readily takes place. The above-mentioned facts can be interpreted within the model for DNA hydrolysis with involvement of two intermediate covalent forms of dNMP residues with DNA polymerase I; one dNMP-intermediate should be placed at the elongation center and the other--at the hydrolysis center. The DNA hydrolysis by 3'----5' exonuclease activity of DNA polymerase I proceeds through these two covalent forms. DNA polymerases alpha from calf thymus and T4 phage do not catalyze the nucleotide exchange between DNA and dNTP from the reaction media.     

Phospholipid metabolism and lysosomal enzyme secretion by leukocytes. Effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP.

The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide.     

Mutations in the S4-H2 loop of eIF4E which increase the affinity for m7GTP

Eukaryotic initiation factor 4E (eIF4E) binds the 5'-cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [gamma-32P]8-azidoguanosine 5'-triphosphate (8-N3GTP) demonstrated cross-linking at Lys-119 in the S4-H2 loop which is distant from the m7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951-961; Friedland et al. (1997) Protein Sci. 6, 125-131]. Modeling studies indicate that 8-N3GTP cross-linked with Lys-119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4-H2 loop produced proteins with a 5-10-fold higher affinity for m7GTP than wild-type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5'-ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.     

Localization of the high-affinity ATP site in adenosine-3':5'-monophosphate-dependent protein kinase type I. Photoaffinity labelling studies with 8-azidoadenosine 5'-triphosphate.

8-Azido-adenosine 5'-triphosphate (n8(3)ATP) appeared to be a suitable photoaffinity label for the protein kinase dependent on adenosine 3':5'-monophosphate (cAMP). It competes with ATP for the high-affinity ATP site in the undissociated form of the kinase and in the phosphotransferase reaction catalyzed by the catalytic subunit. Furthermore, it is accepted as a substrate in the phosphotransfer reaction. n8(3)ATP incorporated into the holoenzyme is covalently bound irradiation. Protection experiments with ATP indicated that this covalent attachment occurs in the high-affinity ATP site of the enzyme. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate shows that n8(3)ATP is bound to the catalytic subunit. After irradiation the enzyme was dissociated by cAMP. Proportional to the incorporated [gamma-32P]n8(3)ATP, a loss in phosphotransferase activity was found. These results support our model that both ATP sites coincide with respect to their adenine binding part. Thus binding of the regulatory subunit to the catalytic subunit would then transform the low-affinity catalytically active ATP site into a high-affinity inactive site.     

Chemico-enzymatic synthesis of an oligonucleotide template for an analog of ACTH (4-10) fragment

Six oligodeoxyribonucleotides ranging from 9-mer to 13-mer were synthesized in solution by the phosphotriester approach and enzymatically joined by T4 DNA ligase. The obtained double-stranded DNA (32 b.p.) with protruding 5'-ends corresponding to the recognition sites for restrictases EcoRI and BamHI represents an oligonucleotide template coding for the modified amino acid sequence 4-10 of the adrenocorticotropic hormone, [Pro8,Gly9,Pro10]ACTH-(4-10).