Studies on the fine structure of puffs in Sciara coprophila

Fine structure of RNA and DNA puffs of Sciara coprophila was studied during late developmental stages of the fourth larval instar. In RNA puffs the predominant structure seen seems to be a diffuse, lampbrush-like thread or threads sectioned in a variety of planes. The thread is composed of filamentous and granular material. Three types of RNA puffs, each with a slightly different morphology, are found. In their development DNA puffs pass through a precise sequence of stages, each with its distinct morphologic and metabolic characteristics. At the initial and final stages, when much of the puff chromatin is in the compacted state, DNA puffs resemble condensed chromosomal bands. In contrast, at stages when most chromatin is diffuse, DNA puffs share many structural characteristics of RNA puffs. Most of the expanded puff area is permeated by lampbrush-like threads composed of fibrils and granules. RNA and DNA puffs were compared with respect to granule size and distribution by means of electron micrographs of known magnification. The results of the statistical analysis show that: 1) The coefficient of variation (C.V.) of the method of measurement falls between 5 and 7%. 2) There is a fluctuation in granule sizes within each puff with a C.V. of 24–26%. 3) The average granule diameter is 238 Å for DNA puffs and 310 Å for RNA puffs; the difference is statistically significant. 4) The variation in mean granule size in a sample of DNA puffs is rather small (C.V. 12%), while the variation in granule size between different RNA puffs is somewhat larger (C.V. 20%). 5) The relative spread of granule sizes in DNA puffs is more restricted than that in RNA puffs. It is evident then that, on the average, DNA puff granules are smaller and more uniform than granules found in RNA puffs.     

Structural changes of dinoflagellate chromosomes by pronase and ribonuclease

When cells of the dinoflagellates Prorocentrum micans and Gyrodinium cohnii are exposed to the proteolytic enzyme pronase or alternatively to ribonuclease, the structure of chromosomes is markedly altered. These changes have been observed electron microscopically in thin sections and spreads. Treatment of cells with pronase removed the bulk of nonfibrillar chromosome material completely unmasking fine chromosomal DNA fibres. Pronase had similar effect also on the dense material which is in contact with chromosomes; fibrillar loops protruding from chromosomes were exposed. However, pronase had no effect on the structural integrity of chromosomes. On the contrary, treatment of cells with ribonuclease loosened the package of chromosomal fibres. Thin sections showed that the tight package of longitudinal periodic structures seen in untreated chromosome was relaxed; chromosome extended longitudinally and formed a linear array of balls. When ribonuclease-treated chromosomes were spread, they were substantially more stretched than untreated chromosomes because of uncoiling of two oppositehanded spiral chromatid bundles. The effect of ribonuclease treatment suggests that unknown RNA species have an important role in the maintenance of permanent condensation of dinoflagellate chromosomes. On the other hand, proteins removable by pronase are also present. Most probably they are not linked to the chromosome structure but represent the matrix of nuclear activity.     

Developmental changes in chromatin of skeletal muscle of rats: acetylation of chromosomal proteins and transcription

In vivo acetylation of chromosomal proteins and RNA synthesis were studied in the skeletal muscle of 3–30 day old developing rats. The levels of acetylated histones and nonhistone chromosomal (NHC) proteins are high at day 3 and decrease as development progresses. Spermine has no significant effect on acetylation. Incorporation of³H-Uridine into RNA of both nuclear and cytoplasmic fractions is also maximum at day 3 and plateaus by day 14. Nuclear RNA synthesis following acetylation of chromosomal proteins is greatly stimulated in all the ages studied, whereas that in cytoplasmic RNA occurs only in 3 day old rats. Such modifications during early development may bring about conformational and functional changes in the chromatin and contribute significantly to the process of terminal differentiation of skeletal muscle cells.     

Distribution patterns of three subfractions of drosophila nonhistone chromosomal proteins: possible correlations with gene activity.

The distribution of three molecular weight subfractions of the Drosophila nonhistone chromosomal proteins (NHC proteins) has been studied using an immunofluorescent technique (Silver and Elgin, 1976). In all three cases, the fluorescence distribution patterns obtained are distinct and reproducible. The results imply that different NHC protein components have different distributions along the polytene chromosomes. A highly selective pattern is obtained using antiserum against subfraction ?; puffs (loci highly active in RNA synthesis) and many nonpuffed chromomeres which are known to puff at other times during the third larval instar or prepupal stages are brightly fluorescent. New RNA synthesis can be induced at 87A, 87B-C1 and other chromomeres by heat shock treatment; these loci, previously stained at low levels, are subsequently stained brightly using the ? serum. The staining of the heat shock puffs appears to be superimposed upon the prior ? pattern. The results suggest that a change in chromosomal structure, as indicated by staining using the ? serum, is associated with gene activity as indicated by puffing. This different chromosomal structure may be the consequence of either a redistribution of a ? antigenic determinant [a new association of specific protein(s) with the active sites] or a change in chromatin configuration [making the ? antigenic determinant(s) newly available to the antibody probe].     

Genome instability in interspecific cell hybrids I. Unstable expression of genes in divergent cell hybrids

Somatic cell hybrids between cells of widely divergent mammalian species display a range of chromosomal and genetic anomalies which may be the equivalent of the “genomic shock” phenomena observed in many plant and animal interspecific hybrids. Mouse-kangaroo hybrids show extreme segregation and fragmentation of the kangaroo chromosomes. Here 1 show that, in addition to the chromosomal instability, some hybrids display unstable expression of three genes borne on the kangaroo active maternal X chromosome. These genes (HPRT, G6PD andPGK) may be co-ordinately inactivated at high frequency, then reactivated once more. I suggest that this reversible inactivation in interspecific hybrids may be the result of an unstable change at an X inactivation centre located in the kangaroo X_q.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.     

Genome-Wide Identification,Biochemical Characterization,and Expression Analyses of the YTH Domain-Containing RNA-Binding Protein Family in Arabidopsis and Rice

RNA-binding proteins are critical to RNA metabolism in cells and, thus, play important roles in diverse biological processes. In the present study, we identified the YTH domain-containing RNA-binding protein (RBP) family in Arabidopsis thaliana and rice at the molecular and biochemical levels. A total of 13 and 12 genes were found to encode YTH domain-containing RBPs in Arabidopsis and rice and named as AtYTH01–13 and OsYTH01–12, respectively. The phylogeny, chromosomal location, and structures of genes and proteins were analyzed. Electrophoretic mobility shift assays demonstrated that recombinant AtYTH05 protein could bind to single-stranded RNA in vitro, demonstrating that the YTH proteins have RNA-binding activity. Analyses of publicly available microarray data, gene expression by qRT-PCR, and AtYTH05 promoter activity indicate that the Arabidopsis AtYTHs and rice OsYTHs genes have distinct and diverse expression patterns in different tissues and developmental stages, showing tissue- and developmental-specific expression patterns. Furthermore, analyses of publicly available microarray data also indicate that many of the Arabidopsis AtYTHs and rice OsYTHs genes might be involved in responses to various abiotic and biotic stresses as well as in response to hormones. Our data demonstrate that the YTH family proteins are a novel group of RBPs and provide useful clues to define their biological functions of this RBP family in plants.     

Identification of Stringent Response-Related and Potential Serological Proteins Released from Bacillus anthracis Overexpressing the RelA/SpoT Homolog,Rsh Bant

RelA and SpoT synthesize ppGpp, a key effector molecule that facilitates the adaptation of bacteria to nutrient starvation and other stresses, known as the stringent response. To investigate the role of Rsh _Bant , a putative RelA/SpoT homolog (encoded by BAS4302) in Bacillus anthracis, we examined the alteration of the secretome profiles after the overexpression of a functional His-Rsh _Bant protein in the B. anthracis strain Sterne at the stationary growth phase. In the ppGpp-deficient E. coli mutant strain CF1693, overexpression of Rsh _Bant restored a ppGpp-dependent growth defect on minimal glucose media. The secretome profiles obtained using a two-dimensional electrophoresis (2-DE) analysis were altered by overexpression of Rsh _Bant in B. anthracis. Among the 66 protein spots differentially expressed >1.5-fold, the 29 proteins were abundant for further identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Functional categorization of those proteins implicated their involvement in various biological activities. Taken together, our results imply that overexpression of a functional His-Rsh _Bant can lead to the increased levels of intracellular ppGpp in B. anthracis, resulting in the significant changes in its secretome profiling. The stringent response-controlled proteins identified are likely useful as potential targets for serodiagnostic applications.     

Identification and characterization of the ATP·Mg-dependent protein phosphatase activator (F A) as a microtubule protein kinase in the brain

The activating factor of ATP·Mg-dependent protein phosphatase (F _A) has been identified in brain microtubules. When using purified MAP-2 (microtubule associated protein 2) and tau proteins as substrates,F _A could phosphorylate MAP-2 to 16 moles of phosphates per mole of protein with aK _m value of 0.4 µM, and tau proteins to 4 moles of phosphates per mole of proteins with aK _m value of about 3 µM. When using microtubules as substrates,F _A could enhance many-fold the endogenous phosphorylation of many microtubule-associated proteins including MAP-2, tau proteins, and several low-molecular-weight MAPs. In contrast to other reported MAP kinases, such as cAMP-dependent protein kinase and Ca⁺²/phospholipid-dependent protein kinase, theF _A-catalyzed phosphorylation of tau proteins could cause an electrophoretic mobility shift on sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that a dramatic conformational change of tau proteins was produced byF _A. Peptide mapping analysis of the phosphopeptides derived from SV8 protease digestion revealed thatF _A could phosphorylate MAP-2 and tau proteins on at least four specific sites distinctly different from those phosphorylated by cAMP-dependent and Ca⁺²/phospholipid-dependent MAP kinases. Quantitative analysis further indicated that approximately 19% of the total endogenous kinase activity in brain microtubules was due toF _A. Taken together, the results provide initial evidence that the ATP·Mg-dependent protein phosphatase activating factor (F _A) is a potent and unique MAP kinase, and may represent one of the major factors involved in phosphorylation of brain microtubules.     

Acid-soluble chromosomal proteins in maize root and callus cells and after rhizogenesis induction in callus tissues

Callus tissues originating fromZea mays root meristem, induced for rhizogenesis callus, meristematic and differentiated maize root cells for isolation of nuclei and acid-soluble chromosomal proteins were used. Cytological investigations proved that rhizogenesis begins with the formation of meristematic centres, followed by root differentiation about 5–12 days after the treatment with α-naphtalene acetic acid (NAA). When applying electrophoresis in 15% polyacrylamide gel, differences between the electrophoretic profiles of acid-soluble chromosomal proteins, isolated from root cells and from callus tissues, were established. The main differences concern histone H1 and probably H4. There are no differences between electrophoretic patterns of acid-soluble chromosomal proteins of nonorganized callus and callus induced for rhizogenesis. The possible explanation of these results is discussed.