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An automated modeling system that represents an object body by a solid model of polyhedral approximation was developed using multi-directional image inputs. The principle of this solid model generation is based on the calculation of viewing pyramids consisting of the image boundary and the corresponding focal point. Applications to the non-contacting three-dimensional measurement of the shape of irregular and complicated object such as a doll, a fish, a spiral shell, and a lemon, to the automated mesh generation for the boundary element analysis of the lemon, to the CAD of a tillage blade, and to the shape, deformation and motion analysis of an aerodynamic tuft, a small tracer particle, and an alcohol wick flame for the flow visualization are presented. It appears to have considerable utility in bio-mechanics and various industrial applications.

For development of a novel method in protein engineering, we studied the use of nonprotein amino acids, namely, amino acids other than the 20 protein-constituting amino acids. We succeeded in in vivo production of human epidermal growth factor (hEGF) substituted with norleucine, an analog of methionine. In the present study we attempted production of hEGF substituted with p-fluorophenylalanine residue (pFPhe), although hEGF does not have Phe residues. We prepared two genes of mutant hEGF; Tyr22 or Tyr29 was replaced by Phe by oligonucleotide-directed mutagenesis. To enhance incorporation of p-fluorophenyl-alanine, these mutant hEGF genes were introduced into an Escherichia coli strain auxotrophic for phenylalanine. The optimum concentration of p-fluorophenylalanine for production of pFPhe-substituted hEGFs was 50 μg/ml. By gel filtration chromatography, and reverse-phase and ion-exchange high performance liquid chromatography, we succeeded in purification of pFPhe-substituted hEGFs. Fluorine substitution of the aromatic ring of each Phe residue probably induces a conformational change of hEGF.

Visualization of a three-dimensional fish behavior based on the successive feature extraction using the time sequential three-dimensional shape modeling is reported. The principle of the modeling is based on the intersected viewing pyramid consisting of each image boundary of the object and the corresponding focal point. Threedirectional video images are simultaneously acquired and sequential solid models are then reconstructed to obtain behavioral parameters such as body shape, skeleton, head, tail, gravity center, direction, attack angle, trajectory, velocity, acceleration, and moving energy. This procedure is entirely automatic requiring no manual operation and appears to have some considerable utility in tracking and measurement of moving objects.

We constructed a new vector containing the promoter and the signal sequence of E. coli phoA gene, the structural gene for the periplasmic alkaline phosphatase. One of the most useful characteristics of this vector is the unique HindIII restriction site located just at the end of the phoA signal sequence. This restriction site was generated by oligonucleotide-directed site-specific mutagenesis without changing the amino acid sequence of the signal peptide. Any kind of foreign structural gene can be easily inserted into the HindIII site by using synthetic oligonucleotides to construct a hybrid gene which has neither an extra sequence nor a deletion between the phoA signal sequence and the foreign structural gene. Human alpha-interferon gene was inserted into this HindIII site. When this hybrid gene was expressed under the control of the phoA promoter region, a low but significant activity was recovered in the cold water wash of the cells after an osmotic shock procedure.

Human insulin-like growth factor II (hIGF-II) was produced inEscherichia coli as a protein fused to human growth hormone. High level expression of the fusion protein was attained with pIBL-1 plasmid. The hIGF-II obtained byin vitro cleavage of the fusion protein with cyanogen bromide was highly purified and its biological activity was assessed.

A RNA molecule has been synthesized that is identical in sequence to Escherichia coli tRNAfMet except that it lacks the base modifications present in the E. coli tRNA. This was achieved by enzymatic joining of chemically synthesized oligonucleotides with chain lengths of 3-10 which were synthesized by the phosphodiester or phosphotriester method. First, quarter molecules of tRNA were constructed by joining of chemically synthesized fragments with RNA ligase. The 5'-quarter molecule (bases 1-20) served as an acceptor in joining reactions with the 3',5'-bisphosphorylated donor molecule (bases 21-34). The 5'-half molecule thus obtained was treated with phosphatase and joined to the 3'-half molecule which was prepared by ligation of the other quarter molecules (bases 35-60, acceptor; bases 61-77, donor) followed by 5'-phosphorylation with polynucleotide kinase. The synthetic tRNA was characterized by oligonucleotide pattern and was partially active in aminoacylation with E. coli methionyl-tRNA synthetase.

Endeavoring to develop a method to biosynthesize proteins substituted with nonprotein amino acids, we attempted the incorporation of L-2-aminohexanoic acid (Ahx) into human epidermal growth factor (hEGF). Escherichia coli YK537 strain harboring plasmid pTA1522, which has the phoA promoter-phoA signal peptide-hEGF gene, was used. Cells were cultured first in high-phosphate medium and then, for induction of the hEGF-encoding gene, transferred to low-phosphate medium containing Ahx (0.25 mg/ml). hEGF and Ahx-substituted hEGF, [Ahx21]hEGF, secreted into the periplasm were recovered. After treatment with H2O2, [Ahx21]-hEGF was clearly separated from methionine-oxidized hEGF by one-step reverse-phase HPLC. Substitution of the methionine residue of hEGF with Ahx was confirmed by the amino acid analysis of [Ahx21]hEGF. The three biological activities of [Ahx21]hEGF were the same as those of hEGF. From the successful production of [Ahx21]hEGF, a basic strategy was established for preparing proteins substituted with nonprotein amino acid (alloprotein). Induction of the phoA promoter of pho regulon and secretion of the product to the periplasm may depress heat shock-like responses and subsequent hydrolysis of the product by cytoplasmic protease.

Oligonucleotides corresponding to the E. coli tRNA (f)(Met) have been synthesized. A diester approach has been used for the synthesis of 6 , 7 , 8 and 9 . A block condensation of nucleotides with 3′-phosphodiesters has been applied to the synthesis of 2 , 3 , 4 and 5 . A new phosphorylating reagent, p-chlorophenyl N-phenylphosphoroamido chloride was synthesized and used for this purpose. Joining of previously synthesized fragments with T4 RNA ligase yielded an eicosanucleotide ( 1 + 2 ) corresponding to bases 1–20 from the 5′-end.

A tuft in the air flow was observed by three CCD video cameras which were installed in the directions nearly perpendicular to each other. In this procedure, a tufted woolen yarn of the diameter of about 1 mm and of the length of 35 mm was attached on the top of a thin post pin.Principle of this shape modelling is that the intersected zone which is constructed from multiple polygonal pyramids, every of which is bounded by the digitized cross sectional area in the picture image of the target and its lens center as a vertex results a polyhedron and becomes a good approximation to that shape if it is convex. This polyhedron is designed to construct a so-called solid model and is described with spatially fixed co-ordinates, so that not only its spatical shape, but also the position, direction, deformation or fluttering at the every moment are able to be estimated.From the reconstructed shape modelling, influence of rigidness due to adhesives near the fixed end and a few millimeters of three dimensional displacement at the free end are observed. Free end moves like a cone of which vertex locates at the middle of the tuft.This method is capable to serve for measurement and analysis of a single tuft characteristics depending on its material, kind of fluid or flow range. And also this has the posibility of quantitative analysis of three dimensional unsteady flow visualization.

A hexanucleotide and an octanucleotide corresponding to tRNAMetf bases 41-46 and 47-54 were synthesized by stepwise addition of mononucleotides. The octanucleotide is the largest oligoribonucleotide synthesized by this method. A pentanucleotide (bases 61-65) and a hexanucleotide (bases 66-71) were synthesized via partially triesterified intermediates. The deblocked products were purified by ion-exchange chromatography and characterized by enzymatic hydrolysis. These oligonucleotides were used as substrates in joining reactions with RNA ligase.

Chemically synthesized fragments corresponding to the 3' end of tRNAfMet from Escherichia coli were joined by T4-induced RNA ligase to yield a heptadecanucleotide (bases 61--77). The 3' terminus of C-C-A was modified by introduction of the ethoxymethylidene group to prevent intra- and intermolecular self-joining reactions at the 3' end. The terminal trimer was phosphorylated using polynucleotide kinase and joined to C-A-A with RNA ligase. The hexamer [C-A-A-C-C-A(ethoxymethylidene)] corresponding to bases 72--77 was obtained in a yield of 60%. An undecanucleotide (bases 61--71) which had been synthesized in a yield of 34% by similar enzymatic joining of U-C-C-G-G to pC-C-C-C-C-G was allowed to react with the 5'-phosphorylated hexamer (bases 72--77) using an excess of RNA ligase to yield the heptadecanucleotide U-C-C-G-G-C-C-C-C-C-G-C-A-A-C-C-A (bases 61--77). The product was identified by homochromatography and nearest neighbor analysis.

Various protecting groups were introduced to the phosphate of 2'-O-benzoyluridine 3'-phosphate. Stability and selective removal of these groups from the trisubstituted phosphate were investigated and nucleotides with trisubstituted phosphate were uesd for the synthesis of dinucleotides by condensation with a properly protected mononucleotide.

A synthetic gene for porcine secretin was ligated with Pst I-cleaved pBR322 by its flanking synthetic Pst I linkers to produce a fused protein consisting of an amino-terminal portion of beta-lactamase and an entire secretin molecule. The hybrid plasmid was transferred into competent Escherichia coli cells. The plasmids, which were proved in our investigation to contain the secretin gene in the desired orientation, were then screened for the production of secretin. This was shown by radioimmunoassay and gel electrophoretic analysis of the polypeptides that were synthesized in E. coli minicells transformed with the hybrid plasmids. Secretin so produced, whose carboxy terminal residue may not be amidated in contrast with natural porcine secretin, showed the same activity in stimulating pancreatic secretion in a bioassay with anesthetized rats as does natural secretin.

P1-Adenosine 5'-P2-o-nitrobenzyl pyrophosphate (nbzlppA) has been synthesized as a substrate for T4 RNA ligase catalyzed 3'-phosphorylation. Incubation of oligoribonucleotides and nbzlppA with RNA ligase yielded oligoribonucleotides having a 3'-o-(o-nitrobenzyl) phosphate. Photochemical removal of the o-nitrobenzyl group provided the free 3'-phosphate. Using [P2-32P] nbzlppA, 3'-termini of oligoribonucleotides could be labelled with 32P. This reaction was applied to modify the 3'-end of donor molecules in joining reaction with RNA ligase. A trinucleotide U-A-G was converted to U-A-Gpnbzl and phosphorylated with polynucleotide kinase. pU-A-Gpnbzl was then joined to an acceptor trinucleotide A-U-G to yield A-U-G-U-A-Gp.

Nascent short DNA chains could result from repair of incorporated uracil residues or be intermediates in discontinuous replication. We have characterized short DNA chains having apyrimidinic/apurinic-sites at 5' ends, the expected intermediates of repair, to distinguish them from RNA-linked replication intermediates. We have synthesized model substrates for the repair products; d(pRib[32P]poly(T)) and d(Rib[32P]poly(T)). Alkaline hydrolysis of both substrates has produced [5'-32P]poly(dT). Nascent short DNA was prepared from an Escherichia coli sof (dut) mutant, in this strain fragments from excision repair of uracil residues accumulate. The products of alkaline treatment are hardly digested by spleen exonuclease which selectively degrades 5'-hydroxyl-terminated DNA. These two results show that alkaline hydrolysis of the uracil repair fragments produces 5'-phosphoryl-terminated DNA, whereas it is known that 5'-hydroxyl-terminated DNA is generated from RNA-linked DNA molecules. The two types of nascent fragments thus can be distinguished by the 5'-terminal structure produced by an alkaline hydrolysis.

The 1H NMR spectrum of human transforming growth factor alpha (TGF-alpha) was analyzed almost completely by the sequential assignment method using two-dimensional NMR techniques. On the basis of the nearly complete sequence-specific resonance assignment, secondary and tertiary structures of human TGF-alpha in solution (pH 4.9, 28 degrees C) were determined to satisfy the upper limits of proton-proton distances derived from nuclear Overhauser effect experiments. Although human TGF-alpha and mouse epidermal growth factor (EGF) share 27% homology in amino acid sequence, the backbone chain folds in the two growth factors are quite similar. The structure and function of TGF-alpha is well characterized by the "mitten model" previously proposed for mouse EGF. The gross shape of the TGF-alpha molecule resembles a mitten. TGF-alpha interacts with the receptor as a mitten would grasp an object. However, there is an appreciable structural difference between the two growth factors in the back of the mitten that is formed by the N-terminal polypeptide segment. This is consistent with the evidence that the backs of these molecules are not involved in the receptor binding.

The Ala-30 and Asn-32 residues involved in the major antiparallel beta-sheet structure of human epidermal growth factor (hEGF) were substituted with various amino acid residues, and the receptor-binding affinities of the nine variant hEGFs were determined by the use of human KB cells. The Ala-30----Arg, Ala-30----His and Ala-30----Phe substitutions drastically reduced the binding affinity, suggesting that the side chain in position 30 of Ala-30 of hEGF is required to be small for the receptor binding. The Asn-32----Asp substitution significantly reduced the binding affinity, while the Asn-32----His variant could bind to the receptor as well as to the wild-type hEGF. Therefore, it seems to be important for receptor binding that the side chain in position 32 does not have a negative charge but does have an NH group. Thus, we propose that, in the ligand-receptor complex, the receptor recognizes, on one side of the antiparallel beta-sheet structure of hEGF, a wider contact area than previously suggested.

A synthetic gene for human epidermal growth factor (hEGF) was joined to a sequence encoding the signal peptide of Escherichia coli alkaline phosphatase. This hybrid gene was placed under the control of the alkaline phosphatase gene (phoA) promoter in a recombinant plasmid, which was used to transfect E. coli. The hybrid protein that was expressed in host cells under conditions of phosphate limitation was processed accurately during the secretion process, and mature hEGF was recovered in the periplasmic fraction. On the other hand, no EGF was detected in the periplasmic space when the synthetic hEGF gene was not accompanied by the phoA signal sequence.

A tetranucleotide C-G-C-Gp (cytidylyl-(3'-5')-guanylyl-(3'-5')-cytidylyl-(3'-5')-guanosine 3'-phosphate) was synthesized by condensation of (Bz) bzC (Bz)-ibG (Bz) p (5) with C (Bz)-ibG (Bz) pNHC6H4S (O) CH3 (6) using 2, 4, 6-triisopropylbenzenesulfonyl chloride. This sequence corresponds to the E. coli tRNAMetf 5'-terminus. A base substituted analog U-G-C-Gp was also synthesized by a similar procedure to that used for C-G-C-Gp. These tetranucleotides were characterized by enzymic hydrolysis.

Synthesis of two oligothymidylic acids, tridecamer and nonadecamer, is described by a rapid and simple solid-phase method on two kinds of polyacrylamide supports derivatized from commercially available Enzacryl Gel K-2. The syntheses were performed by the phosphotriester method using di- and tri-thymidylic acid blocks as the incoming 3'-phosphodiester component. High coupling yields were consistently obtained and the final product was isolated very easily by high performance liquid chromatography on Permaphase AAX.

An eicosanucleotide C--G--C--G--G--G--G--U--G--G--A--G--C--A--G--C--C--U--G--Gp corresponding to the bases 1--20 of the nascent sequence for the Escherichia coli tRNAfMet has been synthesized by the joining of the chemically synthesized oligonucleotides C--G--C--G, G--G--G--U--G--G and A--G--C--A--G--C--C--U--G--Gp using RNA ligase from T4-infected E. coli. The hexanucleotide and decanucleotide were phosphorylated with polynucleotide kinase and [gamma-32P]ATP prior to the joining reactions. The decanucleotide and eicosanucleotide were reconstituted respectively with the 3'-three-quarter molecule obtained by limited digestion with RNase T1 of the natural tRNAfMet from E. coli and the activity of the complex as a methionine acceptor was tested using purified methionyl-tRNA synthetase from E. coli. The amino acid acceptor activity of the reconstituted molecules was 11% and 84% with respect to that of the intact tRNAfMet.

N, N-Dimethyl-p-phenylenediamine, glycine amide and p-methylthioaniline were condensed with uridine 5'-phosphate and the phosphoramidates obtained were tested for their stability in anhydrous pyridine, 50% aqueous pyridine or 80% acetic acid. The p-methylthioanilidate (IIc) was oxidized to give p-methylsulfoxylanilidate of uridine 5'-phosphate (IId) which was found to be 5 times more stable than the p-methylthio compound. The p-methylsulfoxylanilidate of 2'-O-benzoyluridine 3'-phosphate was condensed with the mononucleotide to yield the dinucleotide, MMTrU(OBz)-p-U(OBz)-p in 28% yield.

Two oligonucleotides 14-bases long were synthesized, one complementary to rabbit beta-globin DNA (R beta G14A) and the other with the same sequence except for a single base change (T for C) (R beta G14B). Hybridization conditions were established such that R beta G14A would hybridize to globin DNA while R beta G14B would not. We also synthesized a mixture of 13-base long oligonucleotides (R beta G13Mix), representing eight of the possible coding sequences for amino acids 15-19 of rabbit beta-globin. One of the eight is complementary to globin DNA. R beta G13Mix was found to hybridize specifically to globin DNA under conditions where oligonucleotides forming single base pair mismatches do not. Furthermore, R beta G13Mix was shown to hybridize specifically to colonies containing a plasmid with a globin DNA insert. These results are discussed with respect to a general procedure for screening recombinant clones for those containing DNA coding for a protein of known amino acid sequence.

Synthesis of the milk proteins, casein and α-lactalbumin was not induced in cultured mammary explants from C3H/HeN castrated virgin mice in the presence of lactogenic hormones such as insulin, cortisol and PRL. Replacement therapy with 17-β-estradiol to the castrated mice completely restored the differentiative potentials of mammary explants, inducing synthesis of the two milk proteins. [3H] thymidine incorporation into DNA synthesized in cultured mammary explants was also decreased by castration to less than 50% (P< 0.001) of that obtained in intact mice, and 17-β-estradiol treatment to castrated animals restored DNA synthesis to 90% of the intact level. The addition of insulin to culture medium significantly (P< 0.001) enhanced [3H] thymidine incorporation into DNA in cultured mammary explants from both intact and 17-β-estradiol-treated castrated mice but not from castrated animals. Insulin binding sites (1710 ± 260 sites/cell) to mammary epithelial cells from castrated mice were significantly (P

A synthetic approach to the formylmethionine-tRNA from E. coli is described. The methods employed include 1) phosphodiester synthesis using block condensation or stepwise addition methods, 2) a mixed method combining di- and triester approaches, 3) the phosphotriester method using o-nitrobenzyl for 2′-OH, p-chlorophenyl and anilidate for phosphate and acyl groups for base protection, and 4) joining of the synthetic fragments with RNA ligase.