Total synthesis of a tyrosine suppressor transfer RNA gene. XIV. Chemical synthesis of oligonucleotide segments corresponding to the terminal regions.

Chemical syntheses of the two dodecanucleotides d(T-C-A-A-C-G-T-A-A-C-A-C) and d(A-C-G-T-T-G-A-G-A-A-A-G), the two undecanucleotides d(T-T-T-A-C-A-G-C-G-G-C) and d(T-G-T-A-A-A-G-T-G-T-T), the decanucleotide d(A-G-T-C-C-G-A-A-A-G), and the nonanucleotide d(A-A-T-T-C-T-T-T-C) are described. These deoxyribo-oligonucleotide segments, excluding the decanucleotide, represent the DNA duplex corresponding to the previously determined nucleotide sequence -30 to -51 of the promoter region of the gene for the tyrosine suppressor tRNA (Sekiya, T., Gait, M.J., Norris, K., Ramamoorthy, B., and Khorana, H.G. (1976) J. Biol. Chem. 251, 4481-4489) and include the EcoRI restriction endonuclease sequence at the appropriate 5'-end. The nona- and decanucleotide along with the previously synthesized deoxyribo-oligonucleotide segments 25 to 27 (Ramamoorthy, B., Lees, R.G., Kleid, D., and Khorana, H.G. (1976) J. Biol. Chem. 251, 676-694) together represent the DNA duplex corresponding to the natural nucleotide sequence 121 to 142 of the region adjoining the C-C-A end of the tyrosine tRNA gene and, in addition, a run of nine nucleotides which include the EcoRI restriction enzyme sequence at the 5'-end. The syntheses used protected mono- and oligonucleotides and stepwise condensation methods. A noteworthy feature of the present syntheses was the use of reverse phase high pressure liquid chromatography for the rapid and efficient separation of synthetic reaction mixtures.     

Total synthesis of a tyrosine suppressor tRNA gene. XV. Synthesis of the promoter region.

By use of polynucleotide kinase and polynucleotide ligase, the 10 deoxyoligonucleotide segments, whose syntheses have been described in accompanying papers, have been joined to form the 62-nucleotide-long DNA corresponding to the promoter region of an Escherichia coli suppressor tRNA gene. The following sequence in the joining reactions was used to obtain error-free and optimal yields of the products: 1) joining of Segment P-1 to P-3 in the presence of Segment P-2; 2) joining of Segments P-4 to P-7 to form Duplex [P4-7]; 3) joining of Segments P-8 to P-10 to Duplex [P4-7] to form Duplex [P4-10]; and finally, 4) joining of P-(1 + 3) and P-2 to Duplex [P4-10] to form the total promoter Duplex [P].     

PROPERTIES AND REGIONAL DISTRIBUTION OF TRYPTOPHAN HYDROXYLASE IN THE CHICKEN BRAIN

Tryptophan hydroxylase in young chicken brain had a pH optimum of 7.5–8, depending on the buffer used. It had apparent K_m values for tryptophan and tetrahydrobiopterin of 49 μM and 32 μM respectively. The enzyme in chicken brain, but not rat brain, was cold-shock labile but was stable for up to 4 days at — 20°C. Lability was observed both in tissues and homogenates of these tissues subjected to cold shock, but the extent of loss of activity varied between brain regions. Supernatant fractions did not lose activity after cold shock. The highest level of tryptophan hydroxylase was found in the rostral region of the chicken brainstem. High levels were also found in the caudal region of the brainstem, the midbrain, thalamus, caudate and cerebral cortex. The cerebellum and optic chiasma contained only traces of activity.     

SOLUBLE AND MEMBRANE BOUND CARBONIC ANHYDRASES FROM RAT CNS: REGIONAL DEVELOPMENT

Abstract— The distribution of the soluble, membrane bound and myelin carbonic anhydrase in different regions of the rat CNS was examined as a function of age. A neuraxial progression from spinal cord to upper brain stem was observed for all three enzyme fractions in the 90 day old rat: upper brain stem > lower brain stem and cerebellum > spinal cord. The membrane bound fraction accounted for close to 60% of the total carbonic anhydrase in all regions except the cerebellum where it accounted for only 40%. The developmental pattern of the total membrane bound and soluble fractions were virtually parallel in all regions studied suggesting that they are derived from a common enzyme pool. The myelin enzyme accounts for a small but significant part of the membrane bound fraction and is present at adult levels by 16 days of age indicating it is an early and specific myelin component.     

Changes in brain components during the development of mice homozygous for the locus “dwarf” (dw)1,5

Brain composition and developmental changes were investigated in mice homozygous for the locus dwarf, and characterized by a reduced level of growth hormone, thyroid stimulating hormone, and prolactin, and by secondary hypothyroidism. The difference in adult brain weight (–32%) between the dwarf and the normal mice was not found to parallel the difference in body weight (–71%), whereas the differences in the weight of the liver (–79%) and that of the kidney (–75%) did. Several biochemical parameters of brain development were assayed in dwarf and normal mice between the ages of 15 and 210 days. Levels of cerebrosides, sulfatides, gangliosides, phospholipids, cholesterol, protein, and RNA (per gram wet weight) were the same for the dwarf and the controls, but the net difference in total brain DNA was less than the net total brain RNA difference (–11% vs. –27%). Total brain lipids (absolute quantities) were the same at 15 days. The difference was –37% by the 50th day, and remained constant thereafter. No change in the specific activity of 2,3-cyclic nucleotide 3-phosphohydrolase or 3-phosphoadenosine-5-phosphosulfate: galactocerebroside sulfotransferase was observed. These data suggest that the regulation of the development of brain structures is maintained, but the level of the synthesis of the various brain constituents is reduced in proportion to the brain weight. The development of the dwarf brain seems to proceed harmoniously.Abbreviations used PAPS 3-phosphoadenosine-5-phosphosulfate - PAPS-CST 3-phosphoadenosine-5-phosphosulfate:galactocerebroside sulfotransferase - CNP 2, 3-cyclic nucleotide 3-phosphohydrolase - Neu NAc N-acetylneuraminic acid This paper is part of the Doctorat d'Etat thesis of L. L. Sarliève.     

Admixture estimation using skin reflectance data

Several different methods are suggested for the estimation of admixture proportions in hybrid populations based on skin reflectance data. These methods are applied to hybrid populations of known ancestry and yield results generally quite similar to those expected based on a simple genetic model. Results indicate the usefulness of these methods in hybridization studies and in the development and refinement of models of the genetics of skin color.     

Proteolysis of filament proteins in glial and neuronal cells afterIn vivo stimulation of hippocampal NMDA receptors

An intrahippocampal injection of N-methyl-D-aspartate induced the appearance of degradation products of both the 68 kiloDalton neurofilament protein and the glial fibrillary acidic protein, as revealed by immunoblot techniques. The degradation of these two filament proteins was maximal at 10 days after the lession. The degradation patterns were similar to those induced with calpains or calcium in vitro. There were no degradation effects on the 200 kD neurofilament protein as tested with both mono- and polyclonal antibodies. Consequently, the neuronal degeneration after excessive activation of NMDA receptors appears to involve calcium activation of proteolytic enzymes. The effects on the glial proteins are probably secondary to neuronal damage but could be related to calcium dependent processes.     

The role of cuticle and epidermal cell wall in resistance of rapeseed and mustard to Rhizoctonia solani

A study was conducted to determine whether the cuticles in two genera of the family Cruciferae are effective barriers to infection by Rhizoctonia solani, and whether differences in cuticle and epidermal cell wall thickness and morphology of epicuticular wax exist between resistant and susceptible cultivars. As Canola/rapeseed (Brassica napus) and mustard (Sinapis alba) plants develop from 1 to 3 weeks of age, they become increasingly resistant to R. solani AG2-1 seedling root rot. Seven-day-old seedlings of S. alba cultivars are invariably more resistant than B. napus cultivars. Brassica napus cultivars do not show an obvious cuticle layer at 1 week but at 3 weeks the presence of a cuticle is seen through autofluorescence with a concomitant increase in resistance to R. solani. Removal of the cuticle from 3-week-old hypocotyls by chloroform treatment results in a decrease in cuticular autofluorescence and a significant increase in disease severity in both resistant and susceptible cultivars. Three-week-old plants of S. alba have a much lower percent disease rating and a significantly (p=0.05) thicker cuticle layer than similar-age plants of B. napus. The results suggest that the cuticle plays an important role in the resistance of S. alba and older plants of B. napus to infection by R. solani.     

Dystrophin does not influence regular cytoskeletal architecture but is required for contractile performance in smooth muscle aortic cells.

Cultured vascular smooth muscle cells express distinct histological phenotypes due to a contractile to synthetic stage transition. In this study, we compared the behaviour of cultured aortic smooth muscle cells from young normal and mdx mice. Morphological, immunobiochemical, immunocytochemical analyses and contraction studies of these cells demonstrated that (i) the cell cytoskeleton in mdx mice is not affected by the absence of dystrophin since proteins such as caldesmon, a-actin, and vinculin are expressed similarly in normal mice, (ii) utrophin (or dystrophin-related protein) overexpression does not compensate for the physiological and functional role of the lacking dystrophin. These data suggested that dystrophin and utrophin cannot substitute one another and may play different or complementary roles within smooth muscle cells.