A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue aminoterminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-SerCys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity.Chymotrypsin(ogen) (EC 3.4.21.1) is synthesized and secreted by the exocrine pancreas and, upon delivery to the intestinal tract, is activated by trypsin to form chymotrypsin, a proteolytic enzyme important in the digestive process. In the past 20 years chymotrypsinogen and chymotrypsin have represented prototype molecules for the study of (pro)enzyme structure and function. The complete amino acid sequence of bovine chymotrypsinogen A was determined by Hartley (1) and that for bovine chymotrypsinogen B by Smillie et al. (2). X-ray crystallography structures of the zymogen (3) and the enzyme (4) were determined by Blow and collaborators. Scheele et al. (5) showed that chymotrypsinogen is synthesized as a presecretory protein containing an amino-terminal transport peptide responsible for translocation of the nascent protein across the membrane of the rough endoplasmic reticulum. The partial amino acid sequence of the transport peptide has been recently determined by Came and Scheele (6).In this study we have developed a strategy to correlate the coding potentials of individual cDNAs with specific isoenzymic forms containing topogenic signals responsible for protein translocation across the rough endoplasmic reticulum membrane and export from the cell. T...
Guinea pigs were given radioisotopic 1-methyl-1-nitrosourea (MNU) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) as a single oral dose of 0.1 mmol/kg and uptake into TCA-precipitable material, whole tissue protein and 7-methyl-guanine and O6-methylguanine were determined in brain, kidney, liver, pancreas, small intestine and stomach after 2, 4 and 24 h. The labeling of TCA-precipitable material and the formation of methylated bases by methyl-labelled MNU clearly exceeded that by MNNG in extra-intestinal tissues such as brain, liver, kidney and pancreas. In the stomach, there was greater DNA methylation by MNNG than by MNU. Labelling of TCA-precipitable material and whole tissue protein by the carbonyl group of MNU was greater than that by the guanidino group of MNNG in brain, liver, kidney and pancreas, while MNNG exceeded MNU in the stomach. These data are consistent with the concept that orally administered MNU would be an effective carcinogen in extra-intestinal tissues, while MNNG would be a locally acting gastric carcinogen.
In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.
1-Methyl-1-nitrosourea (MNU) and 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) are carcinogens which methylate nucleic acids and proteins and covalently modify proteins by carbamoylation (MNU) or guanidination (MNNG). Using MNU and MNNG labeled with carbon-14 in the individual carbon positions, the above reactions were quantitated in the isolated guinea pig pancreas, an organ susceptible to tumorigenesis by MNU. Freshly prepared pancreatic lobules were incubated with the labeled drugs (0.03, 0.3 and 1.0 mM) for one hour at 37 degrees C. Alkylated purines from hydrolyzed DNA were separated on Sephadex G10 and acid-soluble nuclear proteins were extracted and separated on polyacrylamide gels. Total uptake of all four labels into lobules was linear with concentration. Acid insoluble radioactivity also increased linearly except for MNNG methylation which plateaued between 0.3 and 1.0 mM. 7-Methylguanine formation by both compounds was approximately ten fold greater than 06-methylation. However, DNA modification by MNU exceeded that by MNNG, especially at the higher drug concentrations. No carbamoylation or guanidination of DNA was detected. Total binding (methylation plus carbamoylation/guanidination) to acid-extractable chromatin proteins was equivalent to DNA modification on a molar basis (approximately 0.35 and 0.08 pmol/microg for exposure to 1.0 mM MNU and MNNG, respectively). All histones were labeled by all drug preparations, with H2A being the principal site of methylation and H2B, H3 and H1 being the major targets of carbamoylation and guanidination. H4 was the least modified histone. Drug binding to cytoplasmic organelles also occurred. These results show a broad spectrum of nuclear and cytoplasmic modification of pancreatic cells by MNU and, to a smaller extent, MNNG.
Three shaved adult male albino rats were trained to press a lever to replace a hot air drive condition (50°C) with either a small reinforcer (32°C) or a large reinforcer (14°C). Following the 10-sec reinforcer, the drive condition was either reinstated immediately (no delay) or after a 15-sec exposure to the drive temperature, during which the bar was withdrawn (delay). Response rate during the no-delay procedure was faster for the small reinforcer than for the large. This relation reversed during the delay procedure. The former observation is similar to a satiation effect and the latter resembles an incentive effect.The effects of thermal stimuli on instrumental behavior have been investigated primarily by students of physiological regulatory systems. Consequently, many traditional questions about the behavioral properties of motivational events have not been asked about thermal stimuli. The present work determines the role of "incentive" in thermally motivated behavior. Incentive is usually identified as an increase in response strength associated with an increase in reinforcement magnitude (Crespi, 1942;Zeaman, 1949;Guttman, 1953).In the thermal equivalent of the reinforcement procedure, a rat is trained to press a bar to terminate a stressful "drive" condition and replace it with a stress-reducing "reinforcer." The reinforcer is terminated automatically and replaced by the drive condition for the next trial. The drive and reinforcer are defined here as stimulus values and not as intervening variables such as thermal stress. Although it might seem that thennal stress could be quantitatively defined as the internal temperature of the animal, it is lamentably true that internal temperature is actually a temperature gradient. A temperature drop of 5°C occurs between the core, which is relatively invariant, and the skin, which is strongly affected by both air temperature and vasotone. The formulation of an internal index of stress requires a greater understanding of the physiology of heat transport than is currently available. For present purposes, it will suffice to specify treatments in terms of air temperature and assume that thermal stress is some function of air temperature.Th e motivational parameters of the thermal reinforcement procedure have been studied with unusually reliable results. As the intensity of either a hot or cold drive stimulus is increased, response rate
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