Fractionation of chromatin by differential solubility in dilute salt.

Chromatin prepared from the livers of rats was fractionated on the basis of solubility in dilute NaCl. Neither of the fractions obtained was enriched in newly synthesized DNA. The salt-soluble fraction had a higher protein content (usually up to 50%) relative to the DNA, and contained 72% or more of the rapidly synthesized RNA. This RNA was found to be complexed with the salt-soluble deoxyribonucleoprotein, not merely co-solubilized with it. Also, polylysine-binding studies showed that about 70% or more of the nucleic acid phosphates were accessible as compared to about 40% in the unfractionated chromatin. These properties suggested that the soluble fraction was enriched in activity transcribed chromatin. In contrast molecular hybridization studies showed that the complexity of the DNA and its homology with cDNA transcribed from rat-liver polysomal mRNA were the same as those of DNA from unfractionated chromatin, or from the salt-insoluble fraction. This suggests that the criteria commonly accepted as distinguishing between euchromatin and heterochromatin in vitro are not invariably valid.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

URIDINE-5-H3 RADIOAUTOGRAPHY OF THE HUMAN SEX CHROMATIN BODY

To obtain an estimate of the rate of RNA synthesis by the heterochromatic sex chromatin body, human female fibroblasts were labeled with uridine-5-H³ and radioautographed. The number of grains over the sex chromatin body was compared with the number of grains over a comparable area of euchromatin. The ratio was 0.37. When corrected for the higher content of DNA per unit area in heterochromatin, the maximum rate of RNA synthesis by the DNA of the sex chromatin body was approximately 18% of the rate of RNA synthesis by a comparable amount of euchromatin DNA. The rate of RNA synthesis by the sex chromatin body did not increase significantly with partial despiralization of this chromatin at prophase.     

Some peculiarities of gene expression in inbred and hybrid rats under normal conditions and after partial hepatectomy

The peculiarities of gene expression in line and hybrid rats in the normalcy and after partial hepatectomy were studied. It was shown that the relationship between the RNA synthesis rate in chromatin and RNA transport in the cytoplasm as well as the correlation between the RNA transport rate and the activity of the protein-synthesizing mechanism vary in animals with different genotypes. After partial hepatectomy the hybrids have their S-period of the cell cycle earlier than the line animals. The hybrids do not differ from the parental forms in the rate of RNA chromatin synthesis; however, the newly synthesized RNA of the hybrids in transported into the cytoplasm 5.7 and 5 times faster than that of the lines Vistar and August. The rapidly transported RNA of hybrids is likely to have a more prolonged life as compared to the line animals. After partial hepatectomy an activation of the whole gene expression system occurs. The differences in the gene expression system of hepatocytes of inbred and hybrid animals in the regenerating liver become less pronounced.     

Quantitative and qualitative aspects of the binding of N-hydroxy-2-acetylaminofluorene to hepatic chromatin fractions

The binding of a chemical carcinogen to components of hepatic chromatin in male rats was examined. After a single injection of N-[3H]hydroxy-2-acetylaminofluorene ([3H]OH-AAF) covalent binding to chromatin RNA, protein, and DNA occurs. The amount of carcinogen bound to RNA was approximately 5 times greater than to DNA, and 10 times that of the protein. However, loss of carcinogen from RNA with time was rapid, whereas a persistent binding to DNA equal to 15% of the initial values was observed. To localize the initial and persistent DNA-bound carcinogen, the genome was fractionated using two different chromatin fractionation procedures. The procedures used yielded 3 chromatin fractions based on physical characteristics, degree of association with nascent RNA and in vitro template capacity. Based on those parameters, these chromatin fractions have been tentatively classified as template expressed euchromatin, a repressed heterochromatin, and a highly condensed pelleted heterochromatin. With both the glycerol gradient chromatin fractionation procedure and the selective MgCl2 chromatin precipitation procedure, the initial (2 h) binding of carcinogen was greatest on the euchromatin DNA. Loss of carcinogen from the DNA, however, was also significantly faster from the euchromatin when compared to the heterochromatin and the pelleted heterochromatin. By 10 days after a single injection of the carcinogen, the largest amount of bound fluorene residues was located on the pelleted heterochromatin DNA, an apparently repressed portion of the genome, while less than 5% of the initial values were found on either the eu- or heterochromatin. When the rats were fed a 2-acetylaminofluorene-containing diet, loss of carcinogen from the pelleted heterochromatin DNA was enhanced, while loss from the euchromatin DNA was reduced. The covalent nature of the carcinogen modification of DNA was confirmed by thin-layer chromatography (TLC). These studies also demonstrated 2 separate carcinogen-purine base adducts which were identified as N-(guanin-8-yl)-N-AF and 3-(guanin-N2-yl)-N-AAF based on either co-chromatography with an authentic standard or on published Rf-values, respectively. The pelleted heterochromatin DNA had a significantly greater proportion of the 3-guanine-N2 adduct when compared to DNA from either the eu- or heterochromatin.     

Diversity of 7 SL RNA from the signal recognition particle of maize endosperm.

An 11 S ribonucleoprotein particle was isolated from maize endosperm and shown to be functionally and structurally equivalent to the mammalian signal recognition particle. However, unlike animal cells which apparently contain a single 7 SL RNA species, maize endosperm contains a heterogeneous population of 7 SL RNA. To investigate this diversity, we have cloned and sequenced a number of the maize endosperm 7 SL RNAs isolated from functionally active SRP preparations. Some maize 7 SL RNAs are strikingly similar, differing by single base changes or short deletions; surprisingly, others share less than 70 percent sequence homology. Despite differences in primary sequence, nearly identical secondary structures can be suggested for all maize 7 SL RNAs, consistent with a proposed functional role in protein translocation for each of these RNAs. The amount of new available sequence data enabled us to define two conserved regions of presumed functional importance: A conserved sequence -G-N-A-R- in the center of a variable region which forms a well defined stem-loop and possibly is involved in an interaction with the 19 kDa protein of the SRP. Secondly, three short nucleotide stretches located in the central domain of 7 SL RNA may form part of a dynamic RNA-switch structure.     

Histone acetylation and chromatin remodeling are required for UV-B-dependent transcriptional activation of regulated genes in maize

The nuclear proteomes of maize (Zea mays) lines that differ in UV-B tolerance were compared by two-dimensional gel electrophoresis after UV light treatment. Differential accumulation of chromatin proteins, particularly histones, constituted the largest class identified by mass spectrometry. UV-B-tolerant landraces and the B73 inbred line show twice as many protein changes as the UV-B-sensitive b, pl W23 inbred line and transgenic maize expressing RNA interference constructs directed against chromatin factors. Mass spectrometic analysis of posttranslational modifications on histone proteins demonstrates that UV-B-tolerant lines exhibit greater acetylation on N-terminal tails of histones H3 and H4 after irradiation. These acetylated histones are enriched in the promoter and transcribed regions of the two UV-B-upregulated genes examined; radiation-sensitive lines lack this enrichment. DNase I and micrococcal nuclease hypersensitivity assays indicate that chromatin adopts looser structures around the selected genes in the UV-B-tolerant samples. Chromatin immunoprecipitation experiments identified additional chromatin factor changes associated with the nfc102 test gene after UV-B treatment in radiation-tolerant lines. Chromatin remodeling is thus shown to be a key process in acclimation to UV-B, and lines deficient in this process are more sensitive to UV-B.     

Rat liver chromatin. Fractionation into eu- and heterochromatin with localization of ribosomal genes.

Purified, normal rat liver chromatin was sheared under controlled conditions in a Virtis “60” homogenizer and then separated into template-active (euchromatin) and template-inactive (heterochromatin) fractions by glycerol gradient centrifugation. The euchromatin portions of the glycerol gradients possessed greater than 90% of the in vitro template activity when estimated with Escherichia coli RNA polymerase and contained more than 90% of the nascent RNA formed in vivo. Inhibitor studies performed in vivo indicated that the leading edge of the heterochromatin portion of the glycerol gradients contains the genes coding for ribosomal RNA. Recentrifugation of putative eu- and heterochromatin fractions demonstrated that the isolation procedure produces fractions which are distinct and are characterized by little or no cross contamination. The data suggest that controlled shearing in conjunction with gradient centrifugation provides a means of fractionating with great fidelity the transcribable portions of the rat liver genome.     

Variations in rat liver chromatin composition during growth and the heterotic response

The manifestation of hybrid vigor with regard to the inheritance of body weight was established as a postweaning phenomenon in rats. Liver weight gain generally corresponded to body weight gain and also reflected hybrid vigor. The protein/DNA ratios of the chromatin from livers of inbred and heterotic hybrid male rats were found to increase with increasing age in growing rats. Hybrid chromatin had higher protein/DNA ratios throughout the age range studies. The difference between hybrids and inbreds were greatest in the early postweaning stages of growth and maturation. The RNA/DNA ratios of chromatin from livers of inbred and hybrid male rats were also found to exhibit trends within certain developmental periods.This investigation was supported by matching Federal and West Virginia State funds from the Hatch 190 grant and is published with the approval of the Director of the Agricultural Station as Scientific Paper No. 1293.     

In vitro transcriptional probes of heterotic rat liver chromatin

DNA-dependent RNA polymerase I from E. coli has been used as probe for determining the capacity of purified chromatin isolated from inbred and hybrid rats during the course of postweaning development to serve as template for RNA synthesis in vitro. An analysis of variance reveals both strain- and age-specific differences in the incorporation of [3H]UTP into RNA. The ability of inbred chromatin to support synthesis exceeds that of the hybrid at every age examined except 50 days, with values in the inbred line approaching those in the hybrid line with increasing age. These data suggest that template characteristics may vary substantially with regard to strain and age and may contribute to differences in heterotic development.     

Kinetics of Heterosis in Growth of the Leaf Blade in Zea mays L.

Early leaf growth in maize inbred parents and their hybrid wasexamined in terms of leaf characters, i.e. length, width, areaof leaf blade, mesophyll cell number, and mesophyll cell size.The vigorous leaf expansion in hybrid can be attributed mainlyto a greater increase in cell number.     

玉米杂种优势与杂交后胚中ATP酶活性和RNA酶活性及蛋白质含量的关系

通过对若干个玉米杂交及其亲本自交授粉后1天、3天、7天、10天胚中ATP酶活性、RNA酶活性及蛋白质变化的比较来探讨杂种优势产生的生化机理。结果表明,30个组合的玉米授粉后一天,有90%组合杂交胚中ATP酶活性超过双亲或母本自交胚中ATP酶活性。授粉后3天杂交胚中ATP酶活性多数低于双亲自交胚中的ATP酶活性,看来这一表现与自交授粉后胚中合子细胞分裂较迟缓有关。以上四个时期授粉后,杂交胚中RNA酶活性介于双亲之间或低于双亲,这可能与杂交胚中核酸分解的速度较慢有关。杂交胚中蛋白质含量变化不同时期各组合间表现不同,杂交后胚胎发育初期胚中蛋白质的变化不一定是总的数量的增加,而是某些蛋白质或酶的变化。     

玉米籽粒贮藏蛋白组成及特性的研究

利用等电聚焦（ＩＥＦ）电泳和不连续醋酸尿素聚丙烯酰胺凝胶电泳（ＮＡＵ－ＰＡＧＥ）对玉米籽粒贮藏蛋白的等电点（ｐＩ）和在Ｆ１中的遗传表现以及贮藏蛋白在胚和胚乳中的分布进行研究,结果表明：（１）玉米籽粒贮藏蛋白的ｐＩ在３．５￣８．４５范围内,可分离的有３９条左右,６０％左右的蛋白质属酸性,ｐＩ分布在３．５０－６．８５范围内,４０％左右属中性偏碱,ｐＩ在６．８５－８．４５范围内。（２）籽粒贮藏蛋白在Ｆ１     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

A cytochemical study of the nonhistone protein content of condensed and extended chromatin

Cytochemical techniques have been used to study the distribution of nonhistone proteins in sections of interphase nuclei and mitotic chromosomes. Condensed chromatin, including the heterochromatin of interphase nuclei from frog liver, and mitotic metaphase and anaphase chromosomes from bovine kidney, show little or no staining for nonhistone protein. Regions of frog liver nuclei which contain extended chromatin (euchromatin) stain intensely for nonhistone protein. These differences in nonhistone staining of condensed and extended chromatin support the suggestion that regions of condensed chromatin contain considerably less nonhistone protein than regions of extended chromatin. The results suggest further that there may be considerably less nonhistone protein associated with chromosomes and interphase heterochromatin than has been reported in most previous analyses of isolated chromatin and chromosome preparations.     

Inheritance of somatic embryogenesis and plantlet regeneration from primary (type 1) callus in maize

Summary Genetic factors controlling the differential expression of somatic embryogenesis and plant regeneration of maize from tissue culture were studied in two crosses. Inbred, hybrid, F2 and backcross generations developed from crossing maize inbred A188 with two commercially important inbred maize lines (B73 and Mo17) demonstrated genetic and environmental effects on somatic embryogenesis and plant regeneration when immature zygotic embryos were cultured on MS medium. Additive gene effects were more important in both crosses than dominant gene effects for precent somatic embryogenesis and percent or number of plants regenerated per embryo when generation means were analyzed. In backcross generations of each cross, cytoplasmic, maternal and/or paternal effects were significant for frequency of somatic embryos three weeks after culture as well as frequency, or number of plants regenerated per embryo, nine weeks after culture. Analysis of genetic variances suggests at least one gene (or block of genes) controls the expression of the frequency of somatic embryogenesis in these crosses. Differences in somatic embryogenesis and plant regeneration between B73 and Mo17 are discussed. This is Journal Paper No. 11,435 of the Purdue University Agricultural Experiment Station.