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A highly efficient stereoselective synthesis of unusual α-silylamines via a direct silyl anion addition reaction is reported. This approach is convergent and avoids any problematic aza-Brook shifts of the anionic intermediates. The use of enantiopure tert-butanesulfinyl imines as the electrophiles affords exceedingly high levels of diastereocontrol for the newly formed stereogenic carbon.

A radio frequency reentrant resonator has been used to determine dielectric constants for one methane + propane mixture and two methane + propane + hexane mixtures along isotherms between 293 K and 313 K, at pressures to 10 MPa. These measurements were made simultaneously with density measurements on the same mixtures, allowing molar polarizabilities to be calculated. Changes in polarizability on mixing were estimated, with component values from literature sources, and were found to average less than 0.6% of mixture polarizability values.

A commercial density/sorption system has been converted to a dual-sinker densimeter and used to make density measurements for some light hydrocarbon mixtures in the compressed gas region and near dew-point conditions. Argon was used as a reference fluid for calibration of the measurement system. Experimental uncertainties in density for methane were estimated as ±(0.029% + 0.013 kg m^-3), including claimed uncertainties in the argon reference data. Measured methane densities at temperatures of (273 to 313) K and pressures to 12 MPa agreed within combined uncertainties with a methane equation of state in the literature (claimed uncertainty of 0.03%). Densities are reported along isotherms in this same range for selected methane + propane and methane + propane + hexane mixtures with cricondentherms below 293 K. Estimated experimental uncertainties for these mixtures are slightly larger than those for pure methane, because of uncertainties in compositions. Additional isochoric experiments were performed on some of these mixtures to investigate the mass adsorbed on each sinker as dew-point conditions are approached. Significant adsorption occurred up to 2 K above the dew point for the binary mixtures and 5 K for the ternary mixtures. Adsorption progressively increased as the dew-point temperature was approached, resulting in changes in gas-phase composition and density. These experiments allowed dew-point conditions and densities to be estimated.

A stainless steel reentrant cavity with primary resonances at three frequencies has been constructed and used to make dielectric constant measurements on liquid water between 300 K and 350 K, at pressures to 13 MPa. At the frequencies of this study (25 MHz to 250 MHz), there is no evidence of frequency dependence in the water data. Three methods have been used to convert resonant frequencies to dielectric constants. Using a lumped-parameter model recommended recently by NIST investigators, the dielectric constants from the current work agree within 0.1 with the best data available from other recent investigations. Temperature and pressure derivatives of the dielectric constant are nearly identical using all three methods, and they are found to be in reasonable but not perfect agreement with the best available correlation of previous results. A waveguide model approach resulted in dielectric constants about 0.3 higher than accepted values, likely due to uncertainties in geometry and materials properties. A second lumped-parameter approach also failed, likely due to its dependence on measured resonant frequency half widths with water occupying the cavity.

The relative potencies of (–)-noradrenaline, (–)-adrenaline, (±)-isoprenaline and (±)-salbutamol have been assessed for their positive inotropic and chronotropic actions on kitten isolated atria. These relative potencies have been compared with those obtained for the relative coronary dilator potencies in two preparations. These were intact hearts perfused by Langendorff’s method and isolated perfused coronary arteries from the kitten. The relative molar potencies for inotropic effects were noradrenaline 1: adrenaline 1: iso-prenaline 7: salbutamol 0.6. The observed ratios for chronotropic effects were not significantly different from those for inotropic effects. The relative potencies of noradrenaline, adrenaline and isoprenaline as myocardial stimulants were similar to their relative potencies as coronary dilators in the intact heart. Similar relative potencies for noradrenaline and isoprenaline were also found in the isolated coronary artery but adrenaline was only one third as active as noradrenaline on this preparation. In both the intact heart and in the isolated coronary artery salbutamol was about one tenth as active as noradrenaline. It was therefore less active relative to noradrenaline as a coronary dilator than as a myocardial stimulant. In spite of these differences in relative potency ratios for myocardial and coronary dilator effects, similar dissociation constants (KBvalues) for practolol against isoprenaline were found in driven atria and in isolated coronary arteries. Myocardial and coronary vascular β-adrenoceptors thus can both be placed in the general classification of β1-adrenoceptors.

Concentration-effect curves to phenylephrine are shifted to the right in the presence of endothelium in rat aorta while responses to clonidine are practically abolished. Analysis of the concentration-effect curves showed that the effect of endothelium could possibly be explained by a reduction in relative intrinsic efficacy of the two agonists by between 3.5- and 6-fold. Published observations on the modulatory effects of agonist-induced contractions by endothelium in the rat aorta tend to support this explanation. It is further concluded that this reduction in efficacy could be related to the basal release of an endothelium-derived substance and that changes in tissue contractility in the presence of endothelium cannot necessarily be taken as evidence for a stimulated liberation of endothelium-derived products by agonists.

In the commonly accepted mechanism for enzymatic hydrolysis of cellulose, endo-beta-1,4-glucanases randomly cleave glucosidic bonds within glucan polymers, providing sites for attack by exo-cellobiohydrolases (EC 3.2.1.91). It has been proposed that hydrolysis by Trichoderma reesei cellobiohydrolase II is restricted to the ends of cellulose polymers because two surface loops cover its active site to form a tunnel. In a closely related endoglucanase, E2 from Thermomonospora fusca, access to the substrate appears to be relatively unhindered because the carboxyl-proximal loop is shortened, and the amino-proximal loop is displaced. The hypothesis was examined by deletion of a region in Cellulomonas fimi cellobiohydrolase A corresponding to part of the carboxyl-proximal loop of T. reesei cellobiohydrolase II. The mutation enhanced the endoglucanase activity of the enzyme on soluble O-(carboxymethyl)cellulose and altered its activities on 2',4'-dinitrophenyl-beta-D-cellobioside, insoluble cellulose, and cellotetraose.

In this paper methodology is described which yields three-way Giemsa differentiation (light-medium-dark) in human metaphase chromosomes exposed to 5-bromode-oxyuridine (BrdU) for 3 DNA synthetic periods (or exposed for 2 DNA synthetic periods and removed from exposure for the third) by means of which all of the sister chromatid exchanges (SCEs) occurring during (or shortly after) S1, S2 and S3 can be accurately counted and distinguished from one another. Using these methods it has been demonstrated that approximately twice as many SCEs occur during the first S-period in the presence of the drug (labeling= B1T0×T0B1)1 as occur during the second S-period (labeling=B2B1× T0B2)1. The three-way differentiation pattern is thought to result from a stepwise decrease in the amount of BrdU incorporated during the first, second and third DNA synthetic periods. These methods can also be used to differentiate between unlabeled (T2T0) and unifilarly labeled (B1T2) sister chromatids and are potentially useful in the detection of sub-chromatid exchanges (none were detected).

Several types of domain occur in beta-1, 4-glycanases. The best characterized of these are the catalytic domains and the cellulose-binding domains. The domains may be joined by linker sequences rich in proline or hydroxyamino acids or both. Some of the enzymes contain repeated sequences up to 150 amino acids in length. The enzymes can be grouped into families on the basis of sequence similarities between the catalytic domains. There are sequence similarities between the cellulose-binding domains, of which two types have been identified, and also between some domains of unknown function. The beta-1, 4-glycanases appear to have arisen by the shuffling of a relatively small number of progenitor sequences.

A cartridge was constructed which contained the divergent tet promoters of transposon Tn10 between an exoglucanase gene (cex) and an endoglucanase gene (cenA) of Cellulomonas fimi. When carried in a broad-host-range vector, the cartridge gave expression of cex and cenA in Escherichia coli, Rhodobacter capsulatus and Klebsiella pneumoniae.

Radioligand binding studies were used to define the binding constants for inhibition of [125I]endothelin-1 binding by endothelin-1 (ET-1), endothelin-3 (ET-3) and a synthetic analogue of ET-1, [Ala1,3,11,15]ET-1 in rat aorta and cerebellum. The inhibition curves for the three substances were similar in the cerebellum (Ki, ET-1 = ET-3 = [Ala1,3,11,15]ET-1) but differed in the aorta (Ki, ET-1 <<< [Ala1,3,11,15]ET-1) with the ET-3 inhibition curves being biphasic, indicating binding to two sites. In functional studies in the aorta, in contrast to findings in the cerebellum, [Ala1,3,11,15]ET-1 was inactive and ET-3 was a partial agonist. These findings support the idea of heterogeneity of endothelin receptors. Autoradiographic studies were performed to identify the cellular localisation of endothelin-1 receptors in the two tissues. In the aorta binding was to all elements of the vasculature including the endothelium, smooth muscle, periadventitial connective tissue and nerves. In the cerebellum binding sites were located predominantly over the neuronal elements of the granular cell layer. The cellular localisation of the high and low affinity sites for ET-3, and the functional significance of the high affinity binding site for ET-3, remain to be determined.

The molecular weight of bacteriophage phi W-14 DNA, determined by velocity sedimentation in neutral sucrose gradients, was 92 +/- 6 X 10(6). The DNA showed marked fragmentation in alkaline sucrose gradients. This fragmentation was not a consequence of preexisting single-strand interruptions in the DNA, since thermal denaturation of DNA yielded intact single strands. The alpha-putrescinylthymine groups in phi W-14 DNA appeared to be labile; some, or parts of some, of these groups were cleaved from the DNA in alkali.

Experiments with a mutant of T4, tsL97, temperature sensitive for gene 43, showed that T4 DNA polymerase was necessary in vivo to repair gaps in recombinant molecules. CsCl density gradient experiments showed that molecular recombinants were not repaired when the T4tsL97-infected cells were shifted to 42 C after replication and recombination had taken place. Repair was almost complete when the same procedure was followed with the wild-type T4, or when the T4tsL97-infected cells were incubated at the permissive temperature, 36 C. Long-single-strand production was also affected similarly by the T4tsL97 mutation. All the results were consistent with the theory that gaps exist in many recombinant molecules at the recombinant joint, that T4 DNA polymerase is the enzyme that repairs these gaps in vivo, and that covalent repair of the recombinants leads to extensive long-single-strand production.

T4 DNA synthesized in a toluene-treated cell system can act as the genetic donor in a DNA transformation assay. This material transforms a variety of markers at high efficiency. We present evidence that the genetic activity is due to newly synthesized, double-stranded DNA.

Three recombinant plasmids, pEC1, pEC2, and pEC3, each containing a unique Cellulomonas fimi chromosomal DNA insert, expressed Cm-cellulase activities in Escherichia coli C600 (Whittle, D. J., Kilburn, D. H., Warren, R. A. J., and Miller, R. C., Jr. (1982) Gene (Amst.) 17, 139-145; Gilkes, N. R., Kilburn, D. G., Langsford, M. L., Miller, R. C., Jr., Wakarchuk, W. W., Warren, R. A. J., Whittle, D. J., and Wong, W. K. R. (1984) J. Gen. Microbiol. 130, 1377-1384). Viscometric and chemical analyses showed that the enzymes encoded by pEC2 and pEC3 behaved as endoglucanases, whereas that encoded by pEC1 behaved as an exoglucanase. The activities of the exoglucanase and the pEC2-encoded endodglucanase were additive on Cm-cellulose as substrate. The pEC1-encoded enzyme also hydrolyzed xylan and p-nitrophenyl cellobioside. Two substrate-bound Cm-cellulases were isolated from the residual cellulose in a C. fimi culture by guanidine hydrochloride elution, affinity chromatography, and polyacrylamide gel electrophoresis. Both were glycoproteins of apparent Mr = 58,000 and 56,000, respectively. The 56-kDa enzyme appeared to be identical with the pEC1-encoded product, suggesting that they arise from the same gene.

Wheat-germ agglutinin is located only in the embryo of a dry wheat seed and not in the endosperm tissue. This distribution remains unaltered for up to 96 h of germination and growth. The lectin is found not only in a freely soluble form but also in reversible association with particulate subcellular components. There appear to be no poly-peptides that can be solubilized with sonication and aqueous buffers from the embryo tissue that can interact with the agglutinin. This suggests that in vivo the lectin remains uncomplexed to endogenous glycoconjugates or is only able to bind to glycosylated integral membrane polypeptides. Alternatively the potential endogenous receptor(s) to wheat-germ agglutinin may not contain a polypeptide. Although the lectin is not present in the endosperm, seven polypeptides able to interact in a reversible way with wheat-germ agglutinin could be purified from that tissue.

Depolarization (100 mM K+) of acetylcholine, serotonin and prostaglandin (PG) F2 alpha contracted human isolated coronary artery segments. Serotonin was about 10-fold more potent than either acetylcholine or PGF2 alpha, but PGF2 alpha elicited contractions of the greatest magnitude. Serotonin (10 microM) and PGF2 alpha (3 microM) induced rhythmic contractions in the arterial segments. Depolarization-, PGF2 alpha- and serotonin-induced increase in tone was depressed by nifedipine, as was the serotonin- and PGF2 alpha -induced rhythmic activity. The induced rhythmic activity was about 100-fold more sensitive to inhibition by nifedipine than was the induced tonic contraction of the arteries. Depolarization-induced contractions and serotonin-induced rhythmic contractions displayed about equal sensitivity to inhibition by nifedipine. Nifedipine was more potent as an inhibitor of depolarization-induced contractions of arteries than it was as an inhibitor of electrically stimulated trabecular strips of ventricle from the same hearts. The inhibition of isolated coronary artery contraction was obtained within the range of nifedipine concentrations observed in the blood of treated patients.

Acetylcholine relaxes rat aorta and increases aortic cyclic GMP levels by a mechanism (or mechanisms) dependent on the endothelium and on extracellular calcium. Therefore, the effects of representatives of different subclasses of calcium entry blockers, verapamil, nifedipine, diltiazem, and bepridil, on maximal acetylcholine (1 microM)-induced increases in cyclic GMP levels were investigated in rat isolated aorta. None of these compounds, at a concentration (3 microM) sufficient to maximally inhibit agonist-stimulated Ca2+ influx into vascular smooth muscle cells, significantly affected either the basal or the acetylcholine-stimulated tissue cyclic GMP levels. On replacing all but 20 mM Na+ by choline, a condition that might be expected to limit or even abolish Na+-Ca2+ exchange, or in the presence of amiloride (1 mM), an inhibitor of Na+-Ca2+ exchange, acetylcholine-stimulated increases in tissue cyclic GMP levels were abolished or inhibited by about 80%, respectively. In choline containing solution acetylcholine relaxant responses were abolished. The presence of amiloride, or the replacement of Na+ by choline, had no effect on increases in cyclic GMP levels evoked by sodium nitroprusside (0.3 microM), an agent that stimulates cyclic GMP formation in smooth muscle without intervention of the endothelium. Replacement of Na+ by Li+ but not the other treatments depressed basal tissue cyclic GMP levels by about 45% but did not abolish either acetylcholine- or sodium nitroprusside-induced relaxant responses. However, the time course of relaxant responses elicited by both these relaxant agonists in precontracted rat aortic rings with endothelium was altered by Li+ replacement; the half-time to relaxation to acetylcholine was increased by about 70-fold. It is concluded that calcium channels, as characterized in smooth muscle and cardiac tissue, are not involved in the stimulated liberation of an endothelial-derived relaxant factor by acetylcholine, but that an Na+-Ca2+ exchange process may be of importance.

The recently identified endothelium-derived peptide, endothelin, is a potent vasoconstrictor, but also binds specifically to many types of smooth muscle and to nerve tissue. Endothelin has been detected in plasma and may have physiological or pathological functions. Like other agonists, endothelin increases the turnover of phosphatidyl inositol and liberates intracellular stocks of Ca2+. It also increases plasmalemmal Ca2+ permeability, an effect that is antagonized by calcium entry blockers in some tissues. However, the characteristics of this antagonism are not always typical of that seen when other types of agonists are employed. It seems that in at least some cell types endothelin might activate specific L-type Ca2+ channels indirectly, perhaps secondarily to the activation of another type of cation channel. The endothelin originally described is one of a family of peptides that are closely related to the sarafotoxins. The comparative pharmacology of these peptides and of some analogues of the originally described endothelin have revealed some surprising differences and may indicate the existence of different endothelin receptors.

Hyperthermia was combined with bleomycin, melphalan and cis-platinum in order to examine cell lethality and oncogenic transformation in C3H10T1/2 cells from the adjuvant use of heat with chemotherapy agents. When cells were exposed concurrently to 42·5°C and each of the three chemotherapy agents, heat enhanced both the cytotoxic and oncogenic potential of the drugs. Hyperthermia-enhanced ratios were largest for bleomycin-treated cells. Examination of transformation incidences expressed as a function of surviving fraction, i.e. the cytotoxicity of treatment and therefore drug-heat efficacy, showed that for a given level of cell killing the combination of heat and cis-platinum resulted in fewer transformants per surviving cell than for cis-platinum alone. Hyperthermia appears to reduce the oncogenic potential of low concentrations of melphalan but has no effect on bleomycin-induced oncogenic transformation.

An endo-beta-1,4-glucanase (CenA) and an exo-beta-1,4-glucanase (Cex) were prepared from Escherichia coli expressing recombinant DNA of the cellulolytic bacterium Cellulomonas fimi. Purification was facilitated by the high affinities of these enzymes for cellulose. An extracellular C. fimi protease cleaved both enzymes in vivo in a highly specific manner. The affinity of the parent enzyme for cellulose was contained independently in an amino-terminal fragment (p20) of CenA and a carboxyl-terminal fragment (p8) of Cex. These fragments contained homologous amino acid sequences which were proposed to comprise cellulose binding domains. Corresponding fragments, p30 from CenA and p35 from Cex, which were unable to bind to cellulose, contained catalytic domains. In both enzymes, the two functional domains were joined by a hinge region consisting solely of prolyl and threonyl residues. The binding domain was excised from CenA by proteolytic cleavage immediately adjacent to the carboxyl terminus of this hinge. Cex was cleaved at an exactly analogous site. p30 and p35 retained several of the catalytic functions of their parent enzymes. However, p30 was less active than intact CenA against microcrystalline cellulose implying a critical role for the binding domain of CenA in the hydrolysis of crystalline substrate.

The gene cbhB from the cellulolytic bacterium Cellulomonas fimi encodes a polypeptide of 1090 amino acids. Cellobiohydrolase B (CbhB) is 1037 amino acids long, with a calculated molecular mass of 109765 Da. The enzyme comprises five domains: an N-terminal catalytic domain of 643 amino acids, three fibronectin type III repeats of 97 amino acids each, and a C-terminal cellulose-binding domain of 104 amino acids. The catalytic domain belongs to family 48 of glycosyl hydrolases. CbhB has a very low activity on CM-cellulose. Viscometric analysis of CM-cellulose hydrolysis indicates that the enzyme is an exoglucanase. Cellobiose is the major product of hydrolysis of cellulose. In common with two other exoglycanases from C. fimi, CbhB has low but detectable endoglucanase activity. CbhB is the second exo-cellobiohydrolase found in C. fimi. Therefore, the cellulase system of C. fimi resembles those of fungi in comprising multiple endoglucanases and cellobiohydrolases.

CenA from Cellulomonas fimi is a β-1,4-endoglucanase that binds tightly to cellulose. X-ray-scattering analyses show that the enzyme is tadpole-shaped: the previously identified catalytic and cellulose-binding domains comprise the head and tail respectively. It appears that this structural and functional organization is common to several cellulases from bacteria and fungi.

A strain of Escherichia coli that leaks cellulase activity encoded by cloned cellulase genes from Cellulomonas fimi was isolated following mutagenesis with nitrosoguani-dine. The strain also leaks several other normally periplasmic enzymes into the culture medium: β-lactamase, alkaline phosphate and RNase, and lyses in minimal medium, as judged by cell viability and the release of β-galactosidase. Growth in broth medium affords protection against lysis. Enzyme leakage is temperature dependent, being maximal at 40°C, at which temperature 40% of total cellulase activity is found in the supernatant.

Field-grown wheat (Triticum aestivum L.) has been used as a developmental system to study the appearance of wheat-germ agglutinin during grain maturation. The lectin appears at the mid-grain growth period (30–34 days post-anthesis) and continues to be synthesised throughout the late stages of maturation and desiccation. An acidic endopeptidase activity, inhibited by pepstatin-phenanthroline is present in extracts of embryo and endosperm throughout maturation. After in-vivo labelling of immature embryos with [35S]methionine for 3 h and extraction in the presence of proteinase inhibitors, immunoprecipitates with anti-wheat-germ agglutinin were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, and found to contain three 35S-labelled polypeptides of Mr 46000, 18000 and 13000. Comparison of two-dimensional tryptic maps of 125I-labelled peptides indicate the three polypeptides are closely related.

Five cellulose-binding polypeptides were detected in Cellulomonas fimi culture supernatants. Two of them are CenA and CenB, endo-beta-1,4-glucanases which have been characterized previously; the other three were previously uncharacterized polypeptides with apparent molecular masses of 120, 95, and 75 kDa. The 75-kDa cellulose-binding protein was designated endoglucanase D (CenD). The cenD gene was cloned and sequenced. It encodes a polypeptide of 747 amino acids. Mature CenD is 708 amino acids long and has a predicted molecular mass of 74,982 Da. Analysis of the predicted amino acid sequence of CenD shows that the enzyme comprises four domains which are separated by short linker polypeptides: an N-terminal catalytic domain of 405 amino acids, two repeated sequences of 95 amino acids each, and a C-terminal domain of 105 amino acids which is > 50% identical to the sequences of cellulose-binding domains in Cex, CenA, and CenB from C. fimi. Amino acid sequence comparison placed the catalytic domain of CenD in family A, subtype 1, of beta-1,4-glycanases. The repeated sequences are more than 40% identical to the sequences of three repeats in CenB and are related to the repeats of fibronectin type III. CenD hydrolyzed the beta-1,4-glucosidic bond with retention of anomeric configuration. The activities of CenD towards various cellulosic substrates were quite different from those of CenA and CenB.

The presence of binding sites for [125I]endothelin-1 and the contractile activities of endothelins (ETs) and sarafotoxin S6b and the endothelin fragment ET(16-21) were investigated in guinea pig trachea. ETs and sarafotoxin S6b (0.1-100 nM) induced potent contractile responses in guinea pig trachea with EC50 values ranging from 1.57 to 12.97 nM. Epithelium removal increased the potencies of ET-1, ET-2 and S6b, but not that of ET-3, and maximal responses to ET-1 and ET-2 were also increased. Effects of epithelium removal were partially mimicked by phosphoramidon (10 microM), an enkephalinase inhibitor, suggesting that enkephalinase (EC.3.4.24.11.) is able to degrade ET-1 and ET-2. ET-3-induced contractions were not affected by phosphoramidon. Autoradiographic studies suggested the presence of at least two specific binding sites for [125]ET-1 in guinea pig airway smooth muscle. The correlation between Kd and EC50 values suggests that the binding sites identified in the airway smooth muscle represent functional receptors for ETs. ET(16-21) and ET(16-21)-NH2 were less potent agonists than the ETs in guinea pig trachea and 10 microM ET(16-21) was unable to inhibit [125I]ET-1 binding in guinea pig airway smooth muscle. Therefore, these results suggest that the C-terminal hexapeptide of ET-1 cannot be used to classify ET receptors in guinea pig trachea.

There is increasing concern for the oncogenic potential of agents used to treat cancer. Hyperthermia is one of the few modalities that does not, of itself, produce transformed foci in vitro. The adjuvant use of heat with X-rays or chemotherapy agents is an interesting approach to increasing the cell-killing potential while decreasing the oncogenicity of combined-modality therapy. Following a priming heat dose in C3H 10T1/2 cells, resistance to cell killing by a second heat dose develops and is maximal by 10 h. This is known as thermotolerance, and can be monitored by the appearance of proteins of specific molecular weight known as heat-shock proteins. By contrast, a priming heat dose does not confer resistance to killing by cis-platinum (cis-DDP). Indeed, heat potentiates the cytotoxicity due to cis-DDP. The interaction is greatest if heat and drug are applied simultaneously, but is still substantial if the drug is applied many hours after heating. The loss of interaction between heat and cis-DDP occurs slowly, but by 48 h, heat and drug act independently. Thermotolerant cells are less sensitive to the induction of transformation by X-rays than previously unheated cells. On the other hand, 48 h after a heat exposure, when cells have regained their normal sensitivity to killing by cis-DDP, their sensitivity to the induction of transformation by cis-DDP has also returned to normal.

The specific transcription of a cloned Drosophila melanogaster tRNAVal4 gene and a tRNASer7 gene by extracts from a homologous embryonic cell line showed lag periods of about 30 min before maximum rates were reached. This lag appeared to represent the time to form an active transcription complex. Thus, when extracts were incubated with template DNA for 30 min at 22 degrees C and stored in the cold, the subsequent transcription rate was linear with time and without a lag. After ultracentrifugation of a preincubated reaction mixture on a sucrose step gradient consisting of 20, 30, 40, and 60% shelves, about 40% of the transcription activity in the extract was found in the 40% shelf. This fraction formed almost exclusively RNA I, the unprocessed tRNA gene transcript, and transcription required only addition of ribonucleoside triphosphates. The rate of formation of RNA by the 40% sucrose fraction was linear against time, with no lag, and linear with the quantity of fraction. The yield of activity isolated on the gradient was directly proportional to the quantity of cloned gene in the preincubation mixture. At a limiting concentration of the gene in the preincubation mixture, the turnover number of the isolated complex was approximately 50 transcripts/gene/h. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of fractions containing the complex still showed many bands, although the complex activity was greatly purified compared to the extract. From the sedimentation behavior of the isolated active transcription complex and from its stability and transcriptional properties, we conclude that the 40% sucrose fraction contains an active transcription complex containing a cloned tRNA gene, RNA polymerase III, and the accessory protein factors required for transcription.

Among 728 early chick embryos, 16- to 18-h incubation, 90 were found to be either partially or completely composed of cells with abnormal chromosome complements (frequency = 12.4 ± 1.2 percent). Of the 90 cytogenetically abnormal embryos examined exactly half (45) contained haploid cells either alone or in addition to diploid and/or polyploid cells. The remaining 45 embryos comprised 19 triploid embryos, 2 tetraploids, 1 presumptive pentaploid, 10 diploid/tetraploid mosaics, 2 diploid/triploid mosaics and 11 embryos classified as aneuploids. The 728 pedigreed embryos examined in this series were produced by 117 broiler type females mated with 64 broiler type and 4 white leghorn type males in 135 mating combinations. Sibship data from these 135 mating combinations are reported and the distribution of aberrations in this series and an additional 42 sibships reported previously (Miller et al., 1971) is discussed.

CenA is an endo-β 1,4-glucanase from the cellulolytic bacterium Cellulomonas fimi. It is a bifunctional enzyme comprising an amino-terminal cellulose-binding domain and a carboxyl-terminal catalytic domain joined by a short sequence of prolyl and threonyl residues (the Pro-Thr box). Additional structural and functional information was revealed by a detailed analysis of the products generated by proteolytic cleavage of a nonglycosylated form of CenA. An extracellular C. fimiprotease attacked nonglycosylated CenA at the junctions between the Pro-Thr box and the two functional domains. A stable “core” peptide (p30), corresponding to the catalytic domain, remained after extensive proteolysis. p30 was resistant to further attack even in the presence of 2-mercaptoethanol plus urea or dithiothreitol, but treatment in the presence of sodium dodecyl sulfate allowed complete fragmentation to small peptides. Stable peptides, identical, or closely related to p30, were generated by α-chymotrypsin or papain. These results indicated that the catalytic domain adopts a tightly folded conformation affording protection from proteolytic attack. In contrast, the cellulose-binding domain showed a relatively loose conformation. Progressive proteolytic truncation from the amino terminus was apparent during incubation with α-chymotrypsin or papain, or with C. fimiprotease under reducing conditions. Affinity for cellulose was retained by products missing up to 64 amino-terminal amino acids. The remaining carboxyl-proximal region of the cellulose-binding domain with affinity (47 amino acids) contained sequences highly conserved in analogous domains from other bacterial endo-β 1,4-glucanases. By analogy with other systems, the properties of the Pro-Thr box are consistent with an elongated conformation. The results of this investigation suggest that CenA has a tertiary structure which resembles that of certain fungal cellulases.