Mario Castañeda

Trypanosoma cruzi ribosomal RNA: internal break in the large-molecular-mass species and number of genes.

The large-molecular-mass ribosomal ribonucleic acid from Trypanosoma cruzi probably contains an internal break. The molecule can be obtained in its intact form or in its two fragments depending on the denaturing agents used for its purification and/or display. This break appears to be an in vivo late processing step rather than a random nucleolytic cleavage during in vitro manipulations. Calculations of mass, from gel electrophoretograms, for the large and small main ribosomal ribonucleic acid species and for the two chains derived from the large species gave values of 1.37, 0.84, 0.70 and 0.57 X 10(6) daltons, respectively. Sedimentation velocity measurements in sucrose gradients and in the analytical ultracentrifuge indicated sedimentation coefficients of 24 and 18 S for the large and small main species, respectively. Saturation hybridization curves showed that the nuclear genome, quantified by chemical analysis, contains about 114 ribosomal ribonucleic acid gene copies.

Genome organization and ploidy number in Trypanosoma cruzi.

Trypanosoma cruzi total DNA was analyzed by DNA:DNA reassociation kinetics. The nonlinear least-squares computer solution could reasonably be fitted to three second-order kinetic components. The highly repetitive, middle repetitive, and single copy components comprised 9, 35 and 49% of the genome, respectively. The single copy sequences showed a kinetic complexity of approximately 4 times that of Escherichia coli and of some 11,000 average-sized structural genes. The repetitive sequences (about 6900) presented the long-period pattern of interspersion with a modal length of 7800 bases. The kinetic complexity of total DNA was compatible with a value of at least a diploid genome per cell.

Age-dependent decrease in the activity of protein-synthesis initiation factors in rat brain

The age-dependent reduction of brain protein synthesis was examined at the initiation step of translation in the rat. Activity of brain initiation factor 2, as well as that of other eukaryotic initiation factors that contribute to the binding of initiator aminoacyl-tRNA to ribosomes, was found to decrease with age and to decline parallel to the decrease in total protein synthesis and brain elongation factor 1 activity. Change in the activity of initiation factors was demonstrated by the results of two different assays which compared favorably with each other. Decreased activity of preparations of initiation factors derived from older animals was observed in: (1) saturating conditions; (2) mixtures of preparations from different ages; and (3) both major cell fractions, i.e. cell sap and microsomes.

Role of Elongation Factor 1 in the Translational Control of Rodent Brain Protein Synthesis

Abstract: The translational control of protein synthesis during early postnatal neural development and aging was examined in the mouse and the rat. The activity of brain elongation factor 1 (bEF‐1) was found to decrease exponentially with age and to decline parallel to the age‐dependent decrease in total protein synthesis in both rodents. This decrement in bEF‐1 activity fell within the range of reported age‐related decreases in protein synthesis in in vitro systems. The factor was present in multiple forms; the lighter species predominated in older animals, whereas the young light form apparently disappeared with increasing age, and was replaced by others arising from the heavy form. Elongation factor 1 derived from young brains functioned as a rate‐limiting component in polypeptide synthesis in previously saturated adult systems. The data suggest that bEF‐1 has an important modulatory effect on total brain protein synthesis.

Stagewise decline in the activity of brain protein synthesis factors and relationship between this decline and longevity in two rodent species.

The activities of brain initiation factor 2 and brain elongation factor 1, which function as rate-limiting in total protein synthesis, and estimations of brain weight were followed during postnatal life in the rat and the mouse. Both activities decreased in parallel while cumulative brain weight increased. Three exponential components were required for the mathematical expression of each of the three processes in semilogarithmic plots against time. The acceleration curves for the activities and tissue weight demonstrated a mirror image symmetry. Within the general pattern of diminution with age, the negative acceleration of the activities and the positive acceleration of the brain weight displayed repeated bursts. The activities of both factors could also be arranged into several regression lines in log/log plots against time. Significantly, in these plots, the regression line calculated for the whole set of data for each factor activity showed that the value of the ratio of the slopes (mouse to rat) was inversely related to the square root of the ratio of species longevity and was in agreement with the power law relating life spans of cells to species longevity (Röhme, Proc. Natl. Acad. Sci. U.S.A., 78 (1981) 5009).

An endonuclease restriction analysis of the ribosomal RNA genes of Trypanosoma cruzi

The location of ribosomal RNA genes in total-nuclear-enriched DNA preparations of Trypanosoma cruzi was analyzed by using restriction endonucleases and the eight cytoplasmic ribosomal RNA species of this organism. Two contiguous SstI DNA fragments of about 9.88 and 1.7 kilobase pairs contained the three large-size ribosomal RNA species, 18 S, beta, and alpha, and three small-size ribosomal RNA species, s1, s2 and s3. The other two small-size ribosomal species, s4 and s5, were located outside the ribosomal RNA cistron and independently of each other. Spacers of a presumed large length, about 20 kilobase pairs or more, hampered the identification of putative adjacent ribosomal RNA cistrons.

Small-size ribosomal RNA species in Trypanosoma cruzi.

Trypanosoma cruzi ribosomal RNA was analyzed electrophoresis. On agarose gels, where both large- and small-size species are grossly fractionable, it revealed two bands in the small-size region. These were similar in size to the mammalian 5.8 S and 5 S species. Increased resolution, however, showed these two bands to be composite. The pseudo 5.8 S band contained three, and the pseudo 5 S two, discretely sized molecules. The ribosomal binding of four of these five novel species is apparently dependent on large ribosomal subunit proteins. One species is hydrogen bonded to the beta species of 24 S ribosomal RNA. The five species were estimated to be 261, 217, 197, 141 and 110 nucleotides long.

Trypanosoma (Schizotrypanum) cruzi: repetitive DNA sequence evolution in three geographically distinct isolates

1. Middle-repetitive DNA sequences were analyzed by molecular hybridization to determine both the extent of complementarity and time of evolutionary divergence between isolates of Trypanosoma cruzi from Argentina, Mexico, or Venezuela. 2. Although molecular hybridizations showed no significant difference between the middle-repetitive DNAs of the Mexican and Venezuelan isolates, there was a 2.7% base pair mismatch in hybrid molecules formed by association of strands from both the Mexican and Argentine isolates. 3. Using the rates for divergence of middle-repetitive DNA in sea urchins, the Mexican and Argentine isolates were estimated to have diverged approximately 20-25 million years ago. 4. Analysis of the kinetics for the DNA hybridizations indicates that only minor amplifications of specific gene sequences or changes in the complexity of the genomes could have occurred during the divergence of the three isolates studied.

Phenetic variation in Trypanosoma (schizotrypanum) cruzi isolates

Abstract 1. 1. Thirteen isoenzyme loci were analyzed electrophoretically for each of 13 isolates obtained from Argentina. Costa Rica, Mexico and Venezuela to determine both genetic distance and time of evolutionary divergence between these populations. 2. 2. The isolates from Costa Rica, Mexico and Venezuela appeared as a very homogeneous group and differed from those from Argentina. 3. 3. The tentative values for polymorphism, heterozygosity and number of alleles per locus were higher for the isolates from Argentina than the corresponding values for the other isolates.

Heterogeneity of protein-synthesis initiation factors in developing and aging rat brain

Protein synthesis initiation factors from rat brain were assayed in vitro through the formation of the initiator Met-tRNA X initiation factor 2 X GTP ternary complex. The initiation factor showed different intrinsic activity with increase in age. This difference was elicited by additions of high-salt ribosomal-wash protein to incubation mixtures that were already saturated with protein preparations from brains of older animals. This qualitative difference was further documented by examining the sensitivity of the activities of the initiation factor to spermidine and to temperature. The sensitivity to these effectors varied with age. By fitting an exponential decay model to data from the temperature experiments, it was possible to demonstrate that the preparations of the initiation factor from older brains behaved as a multicomponent system. The brain preparations from older animals contained at least two subpopulations of initiation factor. The fractions of these two gross subclasses varied, inversely one to the other, with age.