David P. Bloch

Histones of Sperm

In “A Catalog of Sperm Histones” (Bloch, 1969), organisms were grouped into five categories according to the basic proteins of their sperm: a Salmo type containing easily extractable “monoprotamines” (Kossel, 1928) whose sole basic amino acid is arginine, a mammalian type containing a very basic “protaminelike” protein that nevertheless binds strongly to the sperm head and can be extracted only after breaking disulfide linkages (Henricks and Mayer, 1965), a Mytilus type easily extractable and intermediate in composition between the monoprotamines and somatic histones (also designated di- and triprotamines by Kossel, 1928), a Rana type showing no discernible differences from somatic histones, and a crab type perhaps containing histones in the cytoplasm but none in the nucleus.

tRNA-rRNA sequence homologies: evidence for an ancient modular format shared by tRNAs and rRNAs.

Homologies between tRNAs and rRNAs are identified in searches using various combinations of Escherichia coli, yeast, Halobacterium volcanii and bovine mitochondrial sequences. As in previously reported comparisons, the homologies are too frequent and long to be attributed to coincidence, and similar frequencies from inter- and intraspecies comparisons preclude evolutionary convergence as an explanation. In contrast to the earlier studies, patterns in the positioning of the homologies are now described. Graphing the positions of the homologies along orthogonal axes that represent numbers of bases in tRNA and rRNA shows recurring patterns in the alignments. Preferred spacings of integral multiples of 9 bases are found, suggesting a periodicity in the ancestral structure from which the tRNAs and rRNAs were derived. The periodicity also suggests persistence of a modular format in both classes of molecules that survived changes in sequence that occurred during evolution. A model is proposed for the generation of the ancestral molecule and the early evolution of the coding mechanism. Elongation by self-priming and self-templating gave a hairpin with a 9 base stem. Two additional cycles gave a 70-80 base tRNA-like structure. Additional cycles yielded a tandem repeat of this unit, roughly equivalent in size to the combined rRNAs of prokaryotes. The larger RNA would contain the information and materials for generating the smaller RNAs. It is proposed that multiple recombination among such molecules gave composite structures, presumed progenitors of todays t- and rRNAs. The distribution of the conserved domains among todays species argues for the existence of the ancestral molecule prior to divergence of lines leading to the various kingdoms. Their presence in the different nucleic acids suggests the existence of a nucleic acid with multiple functions prior to partitioning of these functions among the nucleic acids that exist today. The occurrence of overlaps, overlays and consensus alignments among the homologies provides the means for identifying contiguous and neighboring conserved regions and holds promise for the reconstruction of the sequence of an ancestral molecule.

Isopycnic banding of chromatin in chloral hydrate gradients.

Abstract Chromatin from Ehrlich ascites tumor cells bands in chloral hydrate gradients at densities ranging from 1.40 to 1.60 g/cm 3 . The total chromatin after recovery from the gradient has a composition similar to that of native chromatin. Chloral hydrate does not dissociate the chromatin into its nucleic acid and protein components. The chloral hydrate is inert toward both the DNA and the histone, as indicated by the lack of influence either on the melting curves of the DNA, or on the electrophoretic patterns of the histones, when these substances are recovered from chromatin treated with chloral hydrate. The resolution of chromatin on the gradients reflects different buoyant densities of the chromatin, and this in turn, different protein to DNA ratios. Extraction of the histones from chromatin obtained from different regions of the gradients reveals differences in prevalence of at least two of the histone species, and provides evidence suggesting that different stretches of DNA in the genome may associate with different histones.

Cytochemistry of the Histones

The histones and protamines were discovered along with the nucleic acids late in the last century by Friedrich Miescher 1. They were part of the “nuclein”, first extracted by Miescher in 1869 from pus cell nuclei obtained from old bandages gotten from the hospitals of Tubingen (Miescher 1897). This hard won material was one of a class of biological substances, including casein and phosphoproteins of egg yolk, which were then considered to be unique because of their acidic properties and phosphorus content. Twenty years later however, as the means were devised by Altmann (1889) for resolving the nuclein into its component parts, and the attributes which had previously aroused the most curiosity were found to reside in the nucleic acid portion, most attention was directed toward this substance and the associated proteins were treated as an unwanted by-product obtained during its isolation.

tRNA-rRNA sequence homologies: A model for the origin of a common ancestral molecule, and prospects for its reconstruction

A model is proposed for the early evolution of the coding mechanism. A primordial RNA embodies the functions of todays nucleic acids in a single molecule. The molecule is generated by successive rounds of self-priming and-templating. After proximity is assured by enclosure in a cell, the functions can be partitioned among more efficient specializel molecules. The prediction of sequence homologies in later forms prompted a search for matches between t- and r-RNAs. These are described. Their distributions offer clues to their origins. The existance of overlapping homologies indicates an approach to the reconstruction of an ancestral molecule.

DNA synthesis in nuclei isolated from Ehrlich ascites tumor cells

Abstract 1. 1. Nuclei isolated from Ehrlich ascites cells can synthesize DNA. Maximum synthesis was attained with all four nucleoside triphosphates at concentrations of 1 · 10−4 M, ATP at 6 · 10−3 M, and Mg2+ at 1.0 · 10−2 M. Ascites DNA polymerase stimulated the reaction. The optimal temperature was 37°, and the optimal pH, maintained with 5 · 10−2 M sodium phosphate, 7.5. The maximum rate of incorporation corresponded to a rate of synthesis of 0.6 % per h (total DNA = 100 %) which is 1 4 the normal in vivo rate. This rate was maintained for short periods, and incorporation corresponded to an increment of DNA of 0.16 % of the total, or 1 60 that expected were synthesizing nuclei to complete a round of DNA synthesis. This figure corresponds to about 1 4 the increase expected were nuclei to complete replicons already initiated. 2. 2. Maximum synthesis was observed when nuclei were supplemented with polymerase derived from a nuclear source, which preferentially utilized native DNA. High rates of synthesis were observed when nuclei were supplemented with preparations derived from a non-nuclear source, that preferred single stranded DNA. However, DNA whose synthesis was stimulated by this enzyme was unstable and selectively degraded on further incubation. Under these conditions, two reactions proceeded independently within the nucleus. The exogenous enzyme appeared to utilize different priming sites, and not to interfere with the reaction carried out by the endogenous enzyme, whose product was stable. Addition of ligase prevented stimulation of synthesis by the exogenous reaction. 3. 3. Heating nuclei destroyed their ability to serve a priming function. This capability was restored by treating with pancreatic endonuclease. The ability of different preparations to carry out synthesis with heated nuclei depended upon the presence of endonuclease in the preparations. The polymerase associated endonuclease differed from pancreatic endonuclease as indicated by the kinetics of response of the heated nuclei to these enzymes. 4. 4. Because of the differences in the response evoked in nuclei by nuclear enzyme, and by the single stranded DNA preferring enzyme obtained from these same cells, it is thought that the latter is not involved in the normal duplication of DNA.

tRNA-rRNA sequence homologies: Evidence for a common evolutionary origin?

SummaryMany tRNAs ofE. coli and yeast contain stretches whose base sequences are similar to those found in their respective rRNAs. The matches are too frequent and extensive to be attributed to coincidence. They are distributed without discernible pattern along and among the RNAs and between the two species. They occur in loops as well as in stems, among both conserved and non-conserved regions. Their distributions suggest that they reflect common ancestral origins rather than common functions, and that they represent true homologies.

DNA and histone synthesis rate change during the S-period in Ehrlich ascites tumor cells.

Data from flow-cytometric analysis of DNA of Ehrlich ascites tumor cells were fitted using non-linear least squares curve fitting routines. Analysis of rates of synthesis from the derived S-period profiles revealed a pattern of changing rates of DNA synthesis during the S-period. Three main peaks are seen whose trough to through periods range from 0 to 16%, 16 to 65%, 65 to 100% of the DNA synthesized during S. The differences between the peak rates and rates in the intervening troughs are small, about 10% of the maximum, but these occur reproducibly. Some differences in the DNA distribution profiles, hence rate profiles, can be seen among samples taken at different times during the day. These are thought to reflect the effects of circadian rhythms, but they are not large enough to obscure the general pattern of rate shifts that occur during the S-period. Analyses of radioactivity of 3H-thymidine pulse labelled cells, sorted across the S-period, were in accord with the results obtained from the DNA distributions. A parallel analysis of DNA and histones showed a correspondence in the timing and direction of shifts in rate for both during the middle part of the S-period.

Evolution of E. coli tRNAIle: evidence of derivation from other tRNAs

Abstract Two E. coli tRNAIle sequences were compared against those of 36 other E. coli tRNAs. tRNAIle 1 was found to bear high similarity with tRNAVal 1 (E = 1.11 x 10-18) while tRNAIle 2 had the greatest match ( E = 3 .4 0 x 10-19) with tRNALysl (E is the expected number of such matches, per search, based on coincidence). These matches, which we consider to represent homologies, extend from base 7 to base 67 in the former and base 7 to the end (76) in the latter pair. These results coupled wim otners on tne lower activity ot isoleucine in reactions postulated to be important in primitive protein synthesis (i.e. esterification reactions and non-enzymatic activation by ATP [1-3]) lead us to propose that isoleucine was included among the proteinaceous amino acids, and received its anticodonic assignment, relatively late in evolution through mutation of tRNAs previously employed for other amino acids.

Evolution ofE. coli tRNATrp

Earlier studies (1) have shown there are direct correlations between the hydrophobicity ranking of most amino acids and their anticodonic nucleotides. However, four anticodonic assignments, i.e. those for Trp, Tyr, Ile and the XGA anticodons for Ser, did not correlate. It was our proposal that this failure to correlate was due to the fact that these assignments were made late, relative to the bulk of the assignments, in evolution through the mutation of existing tRNAs. We have shown (2) thatE. coli tRNAIle 1 and tRNAIle 2 were likely derived from tRNAVal 1 and tRNALys respectively andE. coli tRNATyr was possibly derived fromE. coli 5s rRNA or a common precursor with 5s rRNA (3). The fact that quite high homologies were observed in these comparisons is consistent with the late evolution of the tRNAs in question. We now examine the evolution ofE. coli tRNATrp by comparing its homology with otherE. coli tRNAs. The data suggest a possible evolutionary relationship withE. coli tRNAGly or tRNAArg. The data support the idea of the late assignment of anticodons to Trp.