Human origin recognition complex is essential for HP1 binding to chromatin and heterochromatin organization

The origin recognition complex (ORC) is a DNA replication initiator protein also known to be involved in diverse cellular functions including gene silencing, sister chromatid cohesion, telomere biology, heterochromatin localization, centromere and centrosome activity, and cytokinesis. We show that, in human cells, multiple ORC subunits associate with hetereochromatin protein 1 (HP1) alpha- and HP1beta-containing heterochromatic foci. Fluorescent bleaching studies indicate that multiple subcomplexes of ORC exist at heterochromatin, with Orc1 stably associating with heterochromatin in G1 phase, whereas other ORC subunits have transient interactions throughout the cell-division cycle. Both Orc1 and Orc3 directly bind to HP1alpha, and two domains of Orc3, a coiled-coil domain and a mod-interacting region domain, can independently bind to HP1alpha; however, both are essential for in vivo localization of Orc3 to heterochromatic foci. Direct binding of both Orc1 and Orc3 to HP1 suggests that, after the degradation of Orc1 at the G1/S boundary, Orc3 facilitates assembly of ORC/HP1 proteins to chromatin. Although depletion of Orc2 and Orc3 subunits by siRNA caused loss of HP1alpha association to heterochromatin, loss of Orc1 and Orc5 caused aberrant HP1alpha distribution only to pericentric heterochromatin-surrounding nucleoli. Depletion of HP1alpha from human cells also shows loss of Orc2 binding to heterochromatin, suggesting that ORC and HP1 proteins are mutually required for each other to bind to heterochromatin. Similar to HP1alpha-depleted cells, Orc2 and Orc3 siRNA-treated cells also show loss of compaction at satellite repeats, suggesting that ORC together with HP1 proteins may be involved in organizing higher-order chromatin structure and centromere function

Tissue- and development-specific induction and turnover of hsp70 transcripts from loci 87A and 87C after heat shock and during recovery in Drosophila melanogaster

The haploid genome of Drosophila melanogaster normally carries at least five nearly identical copies of heat-shock-inducible hsp70 genes, two copies at the 87A7 and three copies at the 87C1 chromosome sites. We used in situ hybridization of the cDNA, which hybridizes with transcripts of all five hsp70 genes, and of two 3' untranslated region (3'UTR; specific for the 87A7- and 87C1-type hsp70 transcripts) riboprobes to cellular RNA to examine whether all these copies were similarly induced by heat shock in different cell types of D. melanogaster. Our results revealed remarkable differences not only in the heat-shock-inducibility of the hsp70 genes at the 87A7 and 87C1 loci, but also in their post-transcriptional metabolism, such as the stability of the transcripts and of their 3'UTRs in different cell types in developing embryos and in larval and adult tissues. Our results also revealed the constitutive presence of the heat-shock-inducible form of Hsp70 in a subset of late spermatogonial cells from the second-instar larval stage onwards. We suggest that the multiple copies of the stress-inducible hsp70 genes do not exist in the genome of D. melanogaster only to produce large amounts of the Hsp70 rapidly and at short notice, but that they are specifically regulated in a developmental-stage-specific manner. It is likely that the cost/benefit ratio of not producing or of producing a defined amount of Hsp70 under stress conditions varies for different cell types and under different physiological conditions and, accordingly, specific regulatory mechanisms operating at the transcriptional and post-transcriptional levels have evolved

Developmental regulation and complex organization of the promoter of the non-coding hsrω gene of Drosophila melanogaster

The nucleus-limited large non-coding hsrω-n RNA product of the93D or the hsrω gene of Drosophila melanogaster binds to a variety of RNA-binding proteins involved in nuclear RNA processing. We examined the developmental and heat shock induced expression of this gene by in situ hybridization of nonradioactively labelled riboprobe to cellular transcripts in intact embryos, larval and adult somatic tissues of wild type and an enhancer-trap line carrying the hsrω05241 allele due to insertion of aP-LacZ-rosy + transposon at - 130 bp position of the hsrω promoter. We also examined LacZ expression in the enhancer-trap line and in two transgenic lines carrying different lengths of the hsrω promoter upstream of the LacZ reporter. The hsrΩ gene is expressed widely at all developmental stages; in later embryonic stages, its expression in the developing central nervous system was prominent. In spite of insertion of a big transposon in the promoter, expression of the hsrω05241 allele in the enhancer-trap line, as revealed byin situ hybridization to hsrω transcripts in cells, was similar to that of the wild type allele in all the embryonic, larval and adult somatic tissues examined. Expression of the LacZ gene in this enhancer-trap line was similar to that of the hsrω RNA in all diploid cell types in embryos and larvae but in the polytene cells, the LacZ gene did not express at all, neither during normal development nor after heat shock. Comparison of the expression patterns of hsrω gene and those of the LacZ reporter gene under its various promoter regions in the enhancer-trap and transgenic lines revealed a complex pattern of regulation, which seems to be essential for its dynamically varying expression in diverse cell types

Male sterility associated with overexpression of the noncoding hsrω gene in cyst cells of testis of Drosophila melanogaster

Of the several noncoding transcripts produced by the hsrΩ gene of Drosophila melanogaster, the nucleus-limited > 10-kb hsrΩ-n transcript colocalizes with heterogeneous nuclear RNA binding proteins (hnRNPs) to form fine nucleoplasmic omega speckles. Our earlier studies suggested that the noncoding hsrΩ-n transcripts dynamically regulate the distribution of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments. Here we show that a P transposon insertion in this gene's promoter (at -130 bp) in the hsrΩ05241 enhancer-trap line had no effect on viability or phenotype of males or females, but the insertion-homozygous males were sterile. Testes of hsrΩ05241 homozygous flies contained nonmotile sperms while their seminal vesicles were empty. RNA:RNA in situ hybridization showed that the somatic cyst cells in testes of the mutant male flies contained significantly higher amounts of hsrΩ-n transcripts, and unlike the characteristic fine omega speckles in other cell types they displayed large clusters of omega speckles as typically seen after heat shock. Two of the hnRNPs, viz. HRB87F and Hrp57A, which are expressed in cyst cells, also formed large clusters in these cells in parallel with the hsrΩ-n transcripts. A complete excision of the P transposon insertion restored male fertility as well as the fine-speckled pattern of omega speckles in the cyst cells. The in situ distribution patterns of these two hnRNPs and several other RNA-binding proteins (Hrp40, Hrb57A, S5, Sxl, SRp55 and Rb97D) were not affected by hsrΩ mutation in any of the meiotic stages in adult testes. The present studies, however, revealed an unexpected presence (in wild-type as well as mutant) of the functional form of Sxl in primary spermatocytes and an unusual distribution of HRB87F along the retracting spindle during anaphasetelophase of the first meiotic division. It appears that the P transposon insertion in the promoter region causes a misregulated overexpression of hsrΩ in cyst cells, which in turn results in excessive sequestration of hnRNPs and formation of large clusters of omega speckles in these cell nuclei. The consequent limiting availability of hnRNPs is likely totrans-dominantly affect processing of other pre-mRNAs in cyst cells. We suggest that a compromise in the activity of cyst cells due to the aberrant hnRNP distribution is responsible for the failure of individualization of sperms in hsrΩ05241 mutant testes. These results further support a significant role of the noncoding hsrΩ-n transcripts in basic cellular activities, namely regulation of the availability of hnRNPs in active (chromatin bound) and inactive (in omega speckles) compartments

The many ways of returning to the refrain in Telugu song

A refrain (abbreviated R) is a line in a song repeated after each verse, and often used as the\ua0song’s name. Returns to R are usually high points both melodically and lyrically. E.g., a verse\ua0ending "I feel upon my lips again" makes a smooth lead-in (abbreviated L) to the refrain R =\ua0"A taste of honey" (Scott/Marlow, 1962). We notate this "(I feel upon my lips again) A taste of\ua0honey", and call such patterns (lead-in)refrains or (L)R’s. The song goes R … LR … LR, where\ua0L could change verse to verse. In our examples, R and (L)R are often both full sentences. More\ua0interesting L’s are often phrases, clauses or rather than interjections. A word-prefix L can\ua0transform R.Our main contribution is to point out that (L)R patterns are a striking feature of Telugu (TEL)\ua0song, remarkably various and profuse in both old and new songs, yet little remarked in the\ua0literature as far as we are aware. We give examples from the 15th c. to the 21st. In transcription, a\ua0colon marks long vowels, and M, nasalized ones. Retroflexion is shown by capitalization, and\ua0aspiration by h, also a consonant by itself. Glosses are given, some also /morpheme-wise/.Kannada (KAN) and Tamil (TAM) share features with TEL that help make L(R)’s: fairly free word\ua0order, agglutinative particles, and adjectives and relative clauses preceding the noun. We give\ua0only lone KAN and TAM examples, but expect to find more when we search. Hindi (HIN) shares\ua0fewer features with TEL; perhaps therefore, we have so far looked but found few (L)R s in HIN

EVALUATION OF ANTIDEPRESSANT ACTIVITY OF ETHANOLIC EXTRACT OF DIPTERCANTHUS PROSTRATUS NEES

Objective: The major objective of the study is to evaluate the antidepressant activity of ethanolic extract of Dipteracanthus prostratus Nees using rats. The study also aims to assess the effect of the extract on behavioural pattern of treated rats. Methods: The antidepressant activity of the ethanolic extract of Dipteracanthus prostratus was evaluated using Forced swim test (FST). The effect of the extract on the behavioral pattern was assesssed by an Open field test. The oral doses of 500 mg/kg and 250 mg/kg of the extract were fixed after conducting acute toxicity studies. Imipramine (30 mg/kg) was used as the standard. Results: It was observed from the study that the ethanolic extract of Dipteracanthus prostratus at higher concentration showed significantly (p<0.01) reduction in immobility in forced swim test model of depression after repeated administration for 14 d. The results were comparable to the standard drug Imipramine. However, the extract did not any significant activity after acute administration. Neither the ethanolic extract of Dipteracanthus prostratus nor the standard drug imipramine produced any overt behavioural change or motor dysfunction in the open field test. Conclusion: The present study demands a further detailed investigation on the antidepressant and other possible CNS activities of Dipteracanthus prostrates

Green synthesis of magnesium oxide nanoparticles and their antibacterial activity

1210-1215Nanotechnology has prospects of opening new avenues to fight and prevent diseases using atomic-scale tailoring of materials. As the nano revolution emerges, it is imperative to develop “nano‑naturo” links between nanotechnology and green domains of the nature. The present investigation describes the mangrove Rhizophora lamarckii’s property of synthesizing magnesium oxide nanoparticles . The newly synthesized magnesium oxide nanoparticle morphology is nanohexagonal and spherical. The particles range in dimensions between 20 and 50 nm and are crystalline in nature. The functional groups of the mangrove, amine, and alkane are found to act as reductants and stabilizers. The newly synthesized MgO nanoparticles are found to have potent antibacterial activity

SRSF1 modulates the organization of splicing factors in nuclear speckles and regulates transcription

The mammalian cell nucleus is compartmentalized into non-membranous subnuclear domains that regulate key nuclear functions. Nuclear speckles are subnuclear domains that contain pre-mRNA processing factors and non-coding RNAs. Many of the nuclear speckle constituents work in concert to coordinate multiple steps of gene expression, including transcription, pre-mRNA processing and mRNA transport. The mechanism that regulates the formation and maintenance of nuclear speckles in the interphase nucleus is poorly understood. In the present study, we provide evidence for the involvement of nuclear speckle resident proteins and RNA components in the organization of nuclear speckles. SR-family splicing factors and their binding partner, long non-coding MALAT1 RNA, can nucleate the assembly of nuclear speckles in the interphase nucleus. Depletion of SRSF1 in human cells compromises the association of splicing factors to nuclear speckles and influences the levels and activity of other SR proteins. Furthermore, on a stably integrated reporter gene locus, we demonstrate the role for SRSF1 in RNA polymerase II-mediated transcription. Our results suggest that SR proteins mediate the assembly of nuclear speckles and regulate gene expression by influencing both transcriptional and posttranscriptional activities within the cell nucleus

Rapid, diffusional shuttling of poly(A) RNA between nuclear speckles and the nucleoplasm

Speckles are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic or rather an immobile, perhaps structural, component. Fluorescein-labeled oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent protein chimera of the splicing factor SC35 and allowed to hybridize. Fluorescence correlation spectroscopy (FCS) showed that the mobility of the tagged poly(A) RNA was virtually identical in both speckles and at random nucleoplasmic sites. This same result was observed in photoactivation-tracking studies in which caged fluorescein-labeled oligo(dT) was used as hybridization probe, and the rate of movement away from either a speckle or nucleoplasmic site was monitored using digital imaging microscopy after photoactivation. Furthermore, the tagged poly(A) RNA was observed to rapidly distribute throughout the entire nucleoplasm and other speckles, regardless of whether the tracking observations were initiated in a speckle or the nucleoplasm. Finally, in both FCS and photoactivation-tracking studies, a temperature reduction from 37 to 22°C had no discernible effect on the behavior of poly(A) RNA in either speckles or the nucleoplasm, strongly suggesting that its movement in and out of speckles does not require metabolic energy. © 2006 by The American Society for Cell Biology