Reactions of the aminoacyl-tRNA synthetase-aminoacyl adenylate complex and amino acid derivatives. A new approach to peptide synthesis

Several kinds of dipeptide derivative were shown to be formed by the reactions of the aminoacyl adenylate-aminoacyl-tRNA synthetase complex and amino acid ester or amide. It was shown that the peptide bond could be formed by aminoacyl-tRNA synthetases even in the absence of the ribosome.     

Isolation of restriction enzyme EcoVIII, an isoschizomer of HindIII, produced by Escherichia coli E1585-68

A restriction endonuclease designated EcoVIII, an isoschizomer of HindIII, was isolated from Escherichia coli E1585-68 and purified by dextran-polyethylene glycol (DPG) phase partition, ammonium sulfate precipitation, phospho- and DEAE-cellulose chromatography, and hydroxylapatite chromatography. The purified EcoVIII was stable during the purification procedure and its high specific activity required 10 mM Mg²⁺. Unlike HindIII, Eco VIII exhibited a high specific activity even at low pH (pH 6.3) and showed the highest activity at 48° C. Transformation of purified plasmid DNA from E. coli E1585-68 into K-12 indicated that the EcoVIII gene was carried on a multicopy 4.4-kb miniplasmid. EcoVIII seems to be preferable to HindIII for its production and use because of easier handling of producer cells and a wider range of activity.     

The development of functional neuromuscular junctionsin vitro: An ultrastructural and physiological study

Neuromuscular junctions were formedin vitro between rat spinal cord explants and myotubes. At various intervals after the spinal cord explants were added to the myotube culture (7 hr to 15 days of coculture), the presence of functional neuromuscular junctions was determined by recording miniature endplate potentials (mepps) from the myotubes contacted by a few neurites. Electron microscopical studies were conducted on identified myotubes in which mepps were recorded. Mepps were already found as early as 7 hr after coculture. The fine structure of these newly formed neuromuscular junctions was simple. No synaptic specializations were observed except the presence of a small number of synaptic vesicles in the nerve. The neuromuscular junctions differentiated during the coculture period. Synaptic vesicles formed a cluster at the prejunctional membrane with a localized density in the middle. Basal lamina started to form in 4-day-old cocultures and became continuous in cocultures of 10 days or longer. Clear postjunctional foldings were observed in 15-day-old cocultures. Higher mepp frequencies were correlated with more advanced ultrastructure.     

Relationship between hormone content and autonomy in various autonomous tobacco cells cultured in suspension

Various autonomous cultured tobacco cells including crown gallwere examined for their contents of growth regulators by meansof Avena curvature test, cell-division induction test, and tobaccopith callus test. The crown gall cells derived from cv. Hicks produced auxin andcytokinin in the high levels of 300–500 µg IAA equivalentsand 40–80 µg kinetin equivalents per kg, respectively.The major auxin was identified as indole-3-acetic acid basedon mass spectrometry and gas chromatography. These cells alsoproduced methyl indole-3-acetate as a minor component. One ofthe cytokinins was identified as ribosyl-trans-zeatin by meansof both gas chromatography-mass spectrometry and high performanceliquid chromatography. Auxin and cytokinin activities were not detected in the followingthree suspension cultured tobacco cells: cells requiring neithercytokinin nor auxin derived from the callus of N. tabacum cv.Bright Yellow and cells requiring auxin but not cytokinin derivedfrom the calluses of cv. Bright Yellow and cv. Hicks. Theirauxin and cytokinin contents per kg were less than 1 µgIAA equivalent and less than 0.1 µg kinetin equivalent,respectively. The results obtained in this study indicate that enhanced hormonalcontent is not the only reason for autonomous growth. (Received August 16, 1979; )     

Reconstruction of mandibular defects with revascularized free rib grafts.

Two cases of hemimandibular reconstructions with revascularized free rib grafts are presented. The viability of the transplants was confirmed by bone scans and biopsy, even though the main nutrient vessels providing the intramedullary blood flow were not included in these grafts (and only the periosteal circulation was utilized). The removal of a rib graft without the nutrient vessel eliminates the need for a complicated posterior dissection, close to the costovertebral joint. Revascularized free bone grafts have a greater chance of survival, provide more rapid healing, offer less risk of absorption, and are more resistant to infection than conventional bone grafts.     

Detection of brain-specific gene expression in brain cells in primary culture: a novel promoter assay based on the use of a retrovirus vector.

Promoter activities of the brain-specific genes for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were investigated in brain cells in primary culture with the use of a novel retrovirus vector, pIP200. With this vector, promoter activity can be expressed in terms of beta-galactosidase activity. Differentiation of the primary brain cells to mature glial cells was not affected by treatment with the pIP200 virus vector. The 256-bp 5'-flanking region of the GFAP gene directed astrocyte-specific expression of lacZ. It was silent in fibroblasts, even in multiple copies. The 1.3-kb 5'-flanking region of the MBP gene exhibited strict tissue (oligodendrocyte) specificity under the present assay method but showed some leakiness when integrated into the chromosome in multiple copies. Promoter regions conferring cell type specificity in brain were effectively identified by the present method.     

A cytoskeleton-related gene, uso1, is required for intracellular protein transport in Saccharomyces cerevisiae

The Saccharomyces cerevisiae mutant strains blocked in the protein secretion pathway are not able to induce sexual aggregation. We have utilized the defect of aggregation to concentrate the secretion-deficient cells and identified a new gene which functions in the process of intracellular protein transport. The new mutant, uso1, is temperature sensitive for growth and protein secretion. At the restrictive temperature (37 degrees C), uso1 mutant accumulated the core-glycosylated precursor form of the exported protein invertase in the cells. Ultrastructural study of the mutant fixed by the freeze-substitution method revealed expansion of the nuclear envelope lumen and accumulation of the ER at the restrictive temperature. Abnormally oriented bundles of microtubules were often found in the nucleus. The USO1 gene was cloned by complementation of the uso1 temperature-sensitive growth defect. DNA sequence analysis revealed a hydrophilic protein of 1790 amino acids with a COOH-terminal 1,100-amino acid-long alpha-helical structure characteristic of the coiled-coil rod region of the cytoskeleton-related proteins. These observations suggest that Uso1 protein plays a role as a cytoskeletal component in the protein transport from the ER to the later secretory compartments.     

The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies.

Allelic polymorphism in TCR loci may play an important role in shaping the T cell repertoire and in disease susceptibility. We have used a combination of antibody and sequence analysis to investigate polymorphism in the murine V alpha 11 family. Two different antibodies have been analyzed that recognize particular V alpha 11 family members of the V alpha b and V alpha d haplotypes. One antibody shows J alpha dependency, suggesting a conformational element to the epitope. Investigation of the anti-V alpha 11 staining pattern on different mouse strains indicates that there is a marked influence of MHC haplotype on V alpha 11 selection and that V alpha 11 is preferentially expressed on CD4+ cells. Sequence analysis of V alpha 11 genes from the V alpha a, V alpha b, and V alpha d haplotypes shows two potential regions for the haplotype-specific epitopes. The relatedness of the different V alpha 11 family members from different haplotypes suggests that the V alpha 11.1/11.2 gene duplication is relatively recent, but that V alpha 11.3 separated much earlier. Differences between V alpha 11.3 and V alpha 11.1/11.2 are concentrated in the putative complementarity determining regions (CDR), whereas differences between alleles are not clearly clustered. However, the V alpha 11.1a and V alpha 11.1d alleles differ from V alpha 11.1b and V alpha 11.2b in CDR1. A V alpha 11.2-expressing anti-cytochrome c T cell has the same V-J junction as a V alpha 11.1-bearing cell with a similar fine specificity, indicating that V alpha 11.1b and V alpha 11.2b do not contribute different Ag specificities.     

Isolation and identification of bile salts conjugated with cysteinolic acid from bile of the red seabream, Pagrosomus major.

Bile salts present in gallbladder of wild and cultured red seabream, Pagrosomus major, a marine teleost were analyzed. The bile from wild red seabream was found to contain two previously unknown bile salts along with two known bile salts, taurocholate and taurochenodeoxycholate. Isolation of each bile salt was performed by column chromatography. Fast atom bombardment mass spectra of the unknown bile salts showed the molecular ions (M-H)- of m/z 544 and 528 which are shifted 30 mass units upfield compared to those (m/z 514 and 498) of taurocholate and taurochendeoxycholate, respectively; this is consistent with the presence of cysteinolic acid (mol wt 155) instead of taurine (mol wt 125). Enzymatic hydrolysis of the bile salts released cholic acid and chenodeoxycholic acid, respectively, and an amino acid that was identified as D-cysteinolic acid by direct comparison with an authentic sample. From these results, the bile salts in the bile of wild red seabream were identified as the conjugates of cholic acid and chenodeoxycholic acid with cysteinolic acid. 1H- and 13C-magnetic resonance spectra of the bile salts were also consistent with the proposed structure. The cysteinolic acid conjugates were found only in wild and not in cultured red seabream; this distinction seems to result from differences in dietary cysteinolic acid.