Role of paragenome in development

The concept of paragenome is proposed, which is considered as a transient array of short DNA molecules appearing on the chromosome surface during development for the control of genome. The paragenome consists of printomeres, chronomeres, and phylomeres. Chronomeres and printomeres are obligatory for cells of certain differentiation lineages, but the cells of different lineages differ in the sets of these organelles. Phylomeres are facultative, since they appear only when development is modified. The paragenome is a system governing the chromatin configuration and level of structural genes expression, ensuring the interpretation of positional information by the cells and their differentiation in regulatory morphogenesis, and controlling the development in time. Deciphering of the paragenome will allow realization in future of direct reprogramming of somatic cell nuclei without using stem cells and eggs.     

Role of paragenome in development

The concept of paragenome is proposed, which is considered as a transient array of short DNA molecules appearing on the chromosome surface during development for the control of genome. The paragenome consists of printomeres, chronomeres, and phylomeres. Chronomeres and printomeres are obligatory for cells of certain differentiation lineages, but the cells of different lineages differ in the sets of these organelles. Phylomeres are facultative, since they appear only when development is modified. The paragenome is a system governing the chromatin configuration and level of structural genes expression, ensuring the interpretation of positional information by the cells and their differentiation in regulatory morphogenesis, and controlling the development in time. Deciphering of the paragenome will allow realization in future of direct reprogramming of somatic cell nuclei without using stem cells and eggs.     

Compact structure of ribosomal chromatin in Xenopus laevis.

Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation.     

Effect of single-stranded breaks on the ultrastructural organization and cytochemistry of the chromatin in tumor cells

Single-stranded DNA breaks in Zajdela's ascitic hepatoma and transformed hamster fibroblasts were caused by treating alive cells with 1% dimethylsulfoxide for 2 h or 100 micrograms/ml bleomycin for 5 min and tested by alkaline and neutral DNA elutions. Electron microscopy of thin sections revealed decompaction of the loosened approximately 25 nm globules within diffuse chromatin into thin fibrillar mesh while supranucleosomal structure of the compact chromatin remained untouched. The chromatin enhanced its affinity for cationic dyes and contrast agents. It is concluded that the diffuse chromatin possesses torsional stress of DNA superhelicity and its loosened subunits represent a form for its organization. They probably correspond to the functionally active (dynamic) nucleosomes which display destruction under DNA domain relaxation caused by one-strand breaks.     

On the respective roles of the two proteins encoded by the Bacillus sphaericus 1593M toxin genes expressed in Escherichia coli and Bacillus subtilis

The 3.6 kb HindIII DNA fragment of B. sphaericus 1593M chromosomal DNA bears two genes encoding two polypeptides of 41.9 kDa (protein "42") and 51.4 kDa (protein "51"). DNA fragments carrying only one of these two genes when expressed in E. coli yield products that are inactive towards Culex larvae. The larvicidal activity is recovered when Triton X-100 treated E. coli cells containing each one of the two genes are incubated together. In E. coli these two polypeptides are acting synergistically. The protein "51" appears to be involved in the maturation of protein "42" for expression of the larvicidal activity. In B. subtilis however the toxicity is expressed by cells carrying only the gene coding for protein "42". There is no need of the "51" gene product for the maturation of the "42" polypeptide, suggesting that the maturation is most likely accomplished by host enzymes.     

Predicted functional RNAs within coding regions constrain evolutionary rates of yeast proteins

Functional RNAs (fRNAs) are being recognized as an important regulatory component in biological processes. Interestingly, recent computational studies suggest that the number and biological significance of functional RNAs within coding regions (coding fRNAs) may have been underestimated. We hypothesized that such coding fRNAs will impose additional constraint on sequence evolution because the DNA primary sequence has to simultaneously code for functional RNA secondary structures on the messenger RNA in addition to the amino acid codons for the protein sequence. To test this prediction, we first utilized computational methods to predict conserved fRNA secondary structures within multiple species alignments of Saccharomyces sensu strico genomes. We predict that as much as 5% of the genes in the yeast genome contain at least one functional RNA secondary structure within their protein-coding region. We then analyzed the impact of coding fRNAs on the evolutionary rate of protein-coding genes because a decrease in evolutionary rate implies constraint due to biological functionality. We found that our predicted coding fRNAs have a significant influence on evolutionary rates (especially at synonymous sites), independent of other functional measures. Thus, coding fRNA may play a role on sequence evolution. Given that coding regions of humans and flies contain many more predicted coding fRNAs than yeast, the impact of coding fRNAs on sequence evolution may be substantial in genomes of higher eukaryotes.     

Nucleoprotein hybridization: a method for isolating specific genes as high molecular weight chromatin

We describe a new technique designed to isolate specific eukaryotic genes as native oligonucleosome fragments. The isolation method consists of hybridization of single-stranded termini of chromatin restriction fragments to complementary mercurated DNA probes, followed by isolation of the hybrids by sulfhydryl-Sepharose chromatography. SV40 minichromosomes were used to test the effectiveness of the technique. About 80% of KpnI- or BamHI-restricted and lambda exonuclease treated SV40 minichromosomes hybridized to an appropriate DNA probe after a 12-h hybridization reaction under mild conditions (0.1 M aqueous salt, 37 degrees C, pH 8). When the restricted minichromosomes were mixed with a 15-fold excess of "background" chromatin from sea urchin embryos, nucleoprotein hybridization was able to reisolate the SV40 chromatin to 88% purity with a 63% yield. This represented a 115-fold enrichment of specific genes as chromatin. Results of electron microscopy and polyacrylamide gel electrophoresis indicate that the hybridized SV40 chromatin has not lost the major chromosomal proteins characteristic of SV40 nor acquired significant amounts of protein due to exchange with background chromatin. Our experimental results show that it is currently possible to isolate repeated genes from higher eukaryotes for structural and biochemical study of the proteins involved with gene regulation.     

Chromosomal position effects determine transcriptional potential of integrated mammary tumor virus DNA

We have investigated two mammary tumor virus proviruses that are integrated at different chromosomal sites in the genomes of two clonal isolates of cultured rat hepatoma cells. One of these cell lines, J2.17, expresses MTV² RNA only in the presence of glucocorticoid hormones. In contrast, the proviral genes in the other line, J2.15, fail to be transcribed in the presence or absence of glucocorticoids, despite the fact that the viral genes and the cellular components that mediate hormone responses appear intact and normal. Low-level DNAase I digestion of chromosomes in isolated nuclei reveals that the J2.17 MTV DNA sequences are packaged in chromatin that is highly sensitive to DNAase I attack, whereas the chromatin of the J2.15 provirus is relatively resistant to DNAase I. These results demonstrate that the same genetic element located at two different chromosomal loci within a single cell line can differ in both chromatin structure and gene expression. Analysis of the chromatin structure of the appropriate DNA sequences in uninfected HTC cells suggests that the difference in the chromatin structure of the two proviruses may reflect a “spreading effect”, in which heterologous integrating DNA is packaged into chromatin that is similar in configuration to the surrounding chromatin. Thus, we propose that chromosomal position determines the folding pattern of the newly introduced DNA sequences, and that this pattern in turn determines whether the genes can subsequently be expressed in response to the hormonal inducing signal.     

Chromatin diminution in the parasitic nematodes ascaris suum and parascaris univalens

Chromatin diminution in Parascaris univalens and Ascaris suum undoubtedly represents an interesting case of developmentally programmed DNA rearrangement in higher eukaryotes. It is a complex mechanism involving chromosomal breakage, new telomere addition and DNA degradation, and occurs in all presomatic cells. The process is rather specific with respect to its developmental timing and the chromosomal regions that are eliminated. The functional significance of chromatin diminution still remains an enigma. The fact, however, that single-copy, protein-coding genes are contained in the eliminated DNA demonstrates that in P. univalens and A. suum, there is a qualitative difference between germ-line and somatic genomes, and suggests that chromatin diminution may be used as a "throw-away" approach to gene regulation. We present a hypothesis as to how, during evolution, a partial genome duplication might have been linked to the process of chromatin diminution, in order to provide a selective advantage to parasitic DNA-eliminating nematodes.     

The absence of histone H1 from the chromatin fraction obtained by sonication of calf thymus nuclei under "quasiphysiological" ionic conditions.

The minor chromatin fraction was isolated from the sonicated calf thymus nuclei on the basis of its differential solubility in the "quasiphysiological" salt medium (0.1 M KCl-0.05 M NaCl-l mM MgCl2-1 mM CaCl2). Histone Hl is almost completely absent from this fraction. DNA isolated from this fraction occurs in three discrete low mol. wt. fragments. The fraction of chromatin which lacks histone Hl can also be obtained by two other methods. On of them consists in salt precipitation of the chromatin gel and its subsequent sonication. The second method includes precipitation of the sonicated chromatin gel by salts. In the first case the properties of the chromatin fraction which remains in the supernatant after centrifugation closely resemble those of the original salt-soluble nuclear fraction. The second method yields supernatant fraction also lacking histone Hl but containing heterogeneous DNA. Comparisons were also made of the sonically-solubilized nuclear fractions obtained in the complete salt medium and its mono and divalent cationic constituents.     

Alkylation of isolated chromatin with N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

When isolated chromatin is incubated with the carcinogens N-methyl-N-nitrosourea (MeNU) and N-ethyl-N-nitrosourea (EtNU), DNA and chromosomal proteins become alkylated to increasingly greater extents as the carcinogen concentrations increase. With either MeNU or EtNU, the core and linker DNA of chromatin are alkylated to essentially identical extents. Alkylation of chromatin DNA as well as free DNA is drastically reduced at physiological ionic strengths (e.g. 0.15 M NaCl). The presence of 0.15 M NaCl, on the other hand, enhances alkylation of chromosomal proteins. While EtNU is much less reactive to DNA than MeNU, alkylation of chromosomal proteins relative to that of chromatin DNA has been found to be markedly greater with EtNU than with MeNU. Such a difference in their relative reactivities toward DNA and proteins may be related to the known difference of carcinogenic potency between these N-nitroso compounds.     

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.     

Histone tail-independent chromatin binding activity of recombinant cohesin holocomplex

Cohesin, an SMC (structural maintenance of chromosomes) protein-containing complex, governs several important aspects of chromatin dynamics, including the essential chromosomal process of sister chromatid cohesion. The exact mechanism by which cohesin achieves the bridging of sister chromatids is not known. To elucidate this mechanism, we reconstituted a recombinant cohesin complex and investigated its binding to DNA fragments corresponding to natural chromosomal sites with high and low cohesin occupancy in vivo. Cohesin displayed uniform but nonspecific binding activity with all DNA fragments tested. Interestingly, DNA fragments with high occupancy by cohesin in vivo showed strong nucleosome positioning in vitro. We therefore utilized a defined model chromatin fragment (purified reconstituted dinucleosome) as a substrate to analyze cohesin interaction with chromatin. The four-subunit cohesin holocomplex showed a distinct chromatin binding activity in vitro, whereas the Smc1p-Smc3p dimer was unable to bind chromatin. Histone tails and ATP are dispensable for cohesin binding to chromatin in this reaction. A model for cohesin association with chromatin is proposed.