Nucleotide sequence of a fragment of SV40 DNA that contains the origin of DNA replication and specifies the 5' ends of "early" and "late" viral RNA. III. Construction of the total sequence of EcoRII-G fragment of SV40 DNA.

Limited T1 RNase digestion of subfragments of the SV40 DNA restriction endonuclease fragment EcoRII-G were prepared and analyzed. The fragments were separately labeled with 32P at their 5' terminus and the terminal sequences analyzed with limited snake venom diesterase digestion. The data permitted us to deduce the nucleotide sequence for EcoRII-G. The sequence contains a stretch of 17 A-T base pairs preceding the DNA complementary to the 5' end of "early" message RNA, a stretch of 27 bases with a perfect 2-fold rotational symmetry near the origin of DNA replication and a perfect tandem repeat of 21 nucleotides.     

The effects of serotonergic and adrenergic receptor antagonist on prolactin release in the monkey.

The influence of serotonergic and adrenergic antagonists on serum prolactin levels was studied in ketamine anesthetized monkeys. Methysergide, a serotonergic receptor blocker, at 0.035, 0.1 and 1 mg/kg body weight induced a rapid and transient increase in serum prolactin. Cyproheptadine, another serotonergic receptor blocker, at 0.05, 0.5 and 1 mg/kg induced a rapid and sustained increase in serum prolactin. SQ 10631, a third serotonergic receptor blocker, had a minimal effect on increasing basal prolactin levels even at doses as high as 10 mg/kg. Propranolol, a β adrenergic blocker, at a dose of 5 mg/kg induced a small sustained increase in serum prolactin, while a lower dose (1 mg/kg) had a slight but significant effect. Phentolamine, an α adrenergic receptor blocker, at a dose of 5 mg/kg induced a rapid and transient increase in plasma prolactin while a lower dose (1 mg/kg) had no effect. Phenoxybenzamine, a potent α adrenergic receptor blocker, had only a minimal effect on prolactin release even at doses of 3 and 5 mg/kg. It appears that the time course and extent of prolactin release differs among neural antagonists even within the same biogenic amine system.     

The enthalpy of protolysis of liver alcohol dehydrogenase upon binding nicotinamide adenine dinucleotide.

The binding of NAD+, NADH, and ADP-ribose to horse liver alcohol dehydrogenase has been studied calorimetrically as a function of pH at 25 degrees C. The enthalpy of NADH binding is 0 +/- 0.5 kcal mol-1 in the pH range 6 to 8.6. The enthalpy of NAD+ binding, however, varies with pH in a sigmoidal fashion and is -4.0 kcal mol(NAD)-1 at pH 6.0 and +4.5 kcal mol(NAD)-1 at pH 8.6 with an apparent pKa of 7.6 +/- 0.2. The enthalpy of proton ionization of the group on the enzyme is calculated to be in the range 8.8 to 9.8 kcal mol(H+)-1. In conjunction with the available thermodynamic data on the ionization of zinc-bound water in model compounds, it is concluded that the group with a pKa of 9.8 in the free enzyme and 7.6 in the enzyme . NAD+ binary complex is, most likely, the zinc-bound water molecule. Our studies with zinc-free enzyme provide further evidence for this conclusion. Therefore, the processes involving a conformational change of the enzyme upon NAD+ binding and the suggested mechanism of subsequent quenching of the fluorescence of Trp-314 implicating the participation of an ionized tyrosine group must be re-evaluated in the light of this thermodynamic study.     

Mapping and ordering of fragments of BK virus DNA produced by restriction endonucleases.

A total of 51 restriction sites were recognized within the BK virus genome by the combination of 10 different restriction endonucleases. These sites were mapped and oriented relative to one another as well as to the five fragments generated by the digestion of BK virus DNA with HindIII and EcoRI. The result was a comprehensive physical map suitable for in-depth characterization of the functions of BK virus at the molecular level.     

Polyoxazoline-peptide adducts that retain antibody avidity

Synthetic polymers have long been used to modify various properties of proteins such as activity and solubility. Polyethylene glycol (PEG) has been widely used to form adducts with enzymes and antibodies. In this study, the polyoxazoline family of water-soluble polymers was used to synthesize adducts containing a synthetic peptide recognized by a monoclonal antibody (MAb) directed against human protein C (hPC). This is the first application of direct conjugation of unterminated or "living" polymer to a peptide. The avidity of the antibody for the various adducts was characterized with respect to size and hydrophilicity of methyl- and ethyl-substituted polyoxazoline polymers (POX). Avidity of the adducts was not found to be dependent upon the hydrophilicity and was slightly decreased due to polymer modification. The methyl-POX-peptide adducts were found to be highly water soluble, while the ethyl-POX-peptide adducts showed sporadic problems with aqueous solubility. Because the polymer-peptide adducts retained avidity for the antibody, polyoxazoline polymers may have potential application to protein-adduct chemistry.     

Ribosomal protein L35: identification in spinach chloroplasts and isolation of a cDNA clone encoding its cytoplasmic precursor

We describe the isolation of spinach chloroplast ribosomal protein L35 and characterization of a cDNA clone encoding its cytoplasmic precursor. This protein was only recently identified in ribosomes, but the sequences of four L35 genes have now been reported and confirm its presence in eubacteria, chloroplasts, and cyanelles. Using N-terminal sequence data, oligonucleotides were designed and a cDNA library was screened. The nucleotide sequence of the cDNA clones shows that the spinach L35 protein is encoded as a precursor of 159 residues, comprising a mature protein of 73 residues and a transit peptide of 86 residues. The cleavage site for forming the mature protein is deduced to be Thr-Val-Phe-Ala decreases Ala-Lys-Gly-Tyr. The L35 protein in the photosynthetic organelle of the protozoan Cyanophora paradoxa is encoded in the organelle DNA [Bryant & Stirewalt (1990) FEBS Lett. 259, 273-280]. The corresponding gene has not been found in the chloroplast DNA of a lower plant (liverwort) and two higher plants. Our results demonstrate that the L35 protein in a higher plant (spinach) is encoded in the nucleus. This finding, in light of the endosymbiont hypothesis, suggests an organelle to nucleus transfer of the L35 gene at the evolutionary beginnings of land plants.