ALBERT

Notes: This review will concentrate on certain aspects of the nucleic acids of Entamoeba histolytica. Utilization and synthesis of purines and pyrimidines will initially be briefly discussed, e.g. salvage vs. de novo pathways, uptake studies and recognition of at least 4 transport loci. Data will be presented which show that the distribution and synthesis of RNA (to a lesser extent DNA) in the nucleus is basically the opposite one finds in other eukaryotes, viz. most RNA (ribosomal?) is synthesized (or accumulates) in the peripheral chromatin (functional equivalent of nucleolus?). The DNA is distributed and synthesized primarily throughout the nucleus. It is usually so dispersed that it will not stain with e.g. the standard Feulgen technique, unless the DNA condenses around the endosome (not a nucleolar equivalent) prior to nuclear division. Isolation of rRNA was difficult due, in part, to potent and difficult to inhibit RNase(s), some of which are apparently intimately bound to ribosomal subunits. The 25S (1.3 kDa), 178 (0.8 kDa) and 58 rRNA were recovered after isolation with a high salt SDS-DEP technique. This is the only procedure which enables us to obtain high yields of 258 rRNA: guanidine or guanidinium which permits isolation of intact functional mRNA results in isolation of small amounts of 28 RNA relative to 178 RNA. The 258 RNA is “nicked” (apparently during nuclear processing) and dissociates readily into 1 78 (0.7 kDa) and 168 (0.6 kDa) species, and a more rigidly bound 5.88 species. A small amount of “unnicked” 258 RNA was recovered with guanidine. Two DNA-dcpendent RNA polymerases (I and II) with a pronounced preference for denatured DNA as template were eluted from DEAE-Sephadex in reverse order of what occurs in other eukaryotes, except Physarum polycephalum. This conclusion was based on salt optima and alpha-amanitin sensitivity studies. Initial characterization of DNA isolated with a procedure capable of isolating 〉 100-kbp Leishmania DNA showed that undigested DNA migrates as a broad band between markers 6 and 24 kbp. The persistent recovery of such a “band” by us and Perez-Mutul et al. no larger than ca. 24 kbp (with the exception of 〉48 kbp DNA isolated by Hernandez et al. using an in situ lysis technique which did not include a proteinase) may be due to nicks introduced during isolation; or, perhaps much of the amebal DNA exists in vivo as gene sized fragments. However, preliminary data generated using orthogonal pulse-field agarose gel electrophoresis do suggest that amebal DNA may be in small chromosomes.

Notes: SYNOPSIS. The free amino acids of E. invadens and 4 strains of E. histolytica cultured in the CLG medium have been identified by thin layer chromatography. The chromatogram patterns of 3 strains grown for 72 hr at 37°C (103, K-9 and 200) were nearly identical. Amino acids detected on chromatograms according to Rf values, relative positions on chromatogram plates, and identifying colors using a polychromatic ninhydrin spray were: leucine/isoleucine, tyrosine, valine, alanine, glycine, glutamic acid, lysine/ornithine, histidine, proline, plus very small amounts of arginine and possibly serine, aspartic acid and citrulline/glutamine. Cysteic acid may also be present. The same amino acids were detected on chromatograms using comparable extracts of strain Laredo and E. invadens grown for 6 days at room temperature. However, the patterns were different in that serine, glycine, threonine and especially alanine were present in greater abundance in these latter 2 cell types.These chromatogram patterns were compared with similar analyses of strains Laredo and 200 grown in the modified Shaffer-Frye medium of Reeves.Similar analyses are reported on the basic ingredients of the CLG medium, including the cells and protoplasts of the Bacteroides, the non-multiplying bacterial associate employed in the CLG medium. The chromatogram patterns of the Bacteroides, protoplasts and medium were decidedly different from those of the amebae, thus indicating that adequate separation of bacteria, amebae and medium was accomplished.Analyses of the culture filtrates of all cells analyzed revealed a possible difference between the Laredo strain and the other strains of E. histolytica (103, K-9 and 200) in amino acid utilization. Similar differences were observed between the Laredo strain and E. invadens.

Notes: SYNOPSIS. Entamoeba histolytica grown with H3-thymidine in CLG medium took up tritium into DNase-sensitive material in the nucleus and cytoplasm. The distribution of nuclear activity indicated that the entire nucleus, including the peripheral chromatin, may possess DNA; previous investigators reported DNA only in the endosome.The penicillin-inhibited bacterial associate (Bacteroides sp.) used in the CLG medium incorporated tritium from H3-thymidine into autoradiographically detectable DNase-sensitive material. Autoradiographs of amebae fed bacteria prelabeled with H3-thymidine also revealed some nuclear and cytoplasmic label. Thus, the amount of cytoplasmic label due to ingested, prelabeled bacterial DNA and/or actual biosynthesis of cytoplasmic DNA by the amebae themselves, is not known. Also, at least some of the nuclear DNA of amebae is synthesized from ingested bacteria, or, more likely, from bacterial degradation products.

Notes: SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H3-thymidine. Such reports underscore the need to clarify the metabolic fate of H3-thymidine. This investigation outlines the fate of thymidinemethyl-H3 (TMH3) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH3 revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives.Hence in dividing cells, thymidine-methyl-H3 is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected.Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters.Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH3 revealed limited tritium distribution in all fractions.

Notes: SYNOPSIS. Autoradiographic studies were done which tested the effect of a potent DNA inhibitor, mitomycin C (MC) on the utilization of tritium from exogenous thymidine-methyl-H3 (TMH3) in Entamoeba histolytica grown with Bacteroides sp. in CLG medium. Concentrations of MC (0.0002%) which inhibited growth of amebae by ca. 50%, caused an overall depression of tritium utilization by both associate cell and amebae. However, no reduction in percent cells with nuclear activity was apparent.The effect of MC on utilization of tritium in amebae propagated with Bacteroides which were prelabeled with TMH3 was also studied. The extent of labeling and percent amebae with cytoplasmic label was not appreciably depressed by MC. MC did, however, cause a depression of the percent amebae with nuclear label. This would indicate that the utilization of bacterial DNA products for nuclear DNA (reported in a previous communication) is reduced in the presence of MC. These data on the effect of MC on use of exogenous TMH3 and prelabeled Bacteroides provide some evidence that at least some of the nuclear DNA of amebae can be synthesized from the exogenously supplied isotope.Amebae grown with exogenous TMH3 and resuspended in unlabeled medium for 24–28 hrs. with and without MC had a considerable reduction of the extent of label whether MC was present or not. This suggests that the primary effect of MC is not to degrade DNA.

Notes: SYNOPSIS. Using uridine-5-H3, “long-term” labeling experiments over a 72 hr growth cycle were done with E. histolytica strain K9 grown in CLG medium with penicillin-inhibited Bacteroides. Autoradiographic analysis revealed that tritium occurs primarily in cytoplasm and rarely the nucleus of amebae. The most extensive cytoplasmic activity was observed during the initial 0–24 hr growth period of amebae as compared to later labeling periods.RNase or RNase followed by DNase extracted a large amount but not all label from amebae. These nucleases were least effective during the initial 24 hr period of growth. Thus it appears that tritium from uridine-5-H3 is not highly specific for RNA in amebae. However, the possibility that such label is associated with RNase-resistant RNA cannot be ruled out. More recent cytochemical studies do indicate the presence of RNase-resistant RNA in the cytoplasm of amebae. The activity found in penicillin-inhibited Bacteroides after uridine-5-H3 labeling and their reaction to the various digestive procedures was similar to amebae at corresponding labeling periods. Therefore at least some of the RNase-resistant material present in the cytoplasm of amebae may be derived from the ingested bacteria; this has been further found by appropriate experiments in which amebae were fed prelabeled bacteria.Nuclear activity when observed (always after 24 hrs growth) was associated either with the periphery of the nucleus and/or the endosome. It was not seen in the nuclear stroma. Some of this activity is RNase-resistant, perhaps representing double or multi-stranded RNA. It therefore appears that RNA is not distributed in the nuclear stroma in “long-term” labeling experiments.