Characterization of messenger-like ribonucleic acid from Saccharomyces cerevisiae by the use of chromatography on methylated albumin-kieselguhr.
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ABSTRACT:
SUBMITTER: Johnson R
PROVIDER: S-EPMC1179456 | biostudies-other | 1970 Oct
REPOSITORIES: biostudies-other
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Project description:1. Native DNA from two strains of Bacillus subtilis was chromatographed by stepwise elution from MAK (methylated albumin on kieselguhr). 2. Transforming activity was confined to two out of the three main fractions, activity being distributed between the two peaks differently for DNA from the different strains. 3. Fractionation of DNA from both strains on 2% agarose gel gave two components. Approx. 75% of the material was eluted within the void volume of the column. Approx. 25% of the material consisted of degradation products of lower molecular weight. 4. Chromatography on MAK of the material of high molecular weight eluted from agarose gel gave a number of peaks differing in molecular weight, indicating that degradation of the DNA takes place during chromatography on MAK. 5. The distribution of transforming activity among the fractions from MAK suggests that degradation occurs preferentially in certain regions of the DNA.
| S-EPMC1166155 | biostudies-other
Project description:Most of the rapidly labelled RNA from rat liver submitted to column chromatography on methylated albumin on kieselguhr remains tightly bound to the column and can only be recovered by elution with m-ammonia. The tightly bound RNA is composed mainly of DNA-like RNA. The binding capacity is dependent not only on base composition but also on molecular size: the heavier RNA molecules show a greater affinity to the column than do the lower-molecular-weight components. Rapidly labelled mouse liver and Saccharomyces cerevisiae RNA show similar behaviour to rat liver RNA on columns of methylated albumin on kieselguhr.
| S-EPMC1185400 | biostudies-other
Project description:Introduction: Case-control analyses have shown BARD1 variants to be associated with up to >2-fold increase in risk of breast cancer, and potentially greater risk of triple negative breast cancer. BARD1 is included in several gene sequencing panels currently marketed for the prediction of risk of cancer, however there are no gene-specific guidelines for the classification of BARD1 variants. We present the most comprehensive assessment of BARD1 messenger RNA splicing, and demonstrate the application of these data for the classification of truncating and splice site variants according to American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines. Methods: Nanopore sequencing, short-read RNA-seq (whole transcriptome and targeted), and capillary electrophoresis analysis were performed by four laboratories to investigate alternative BARD1 splicing in blood, breast, and fimbriae/ovary related specimens from non-cancer affected tissues. Splicing data were also collated from published studies of nine different tissues. The impact of the findings for PVS1 annotation was assessed for truncating and splice site variants. Results: We identified 62 naturally occurring alternative spliced BARD1 splicing events, including 19 novel events found by next generation sequencing and/or reverse transcription PCR analysis performed for this study. Quantitative analysis showed that naturally occurring splicing events causing loss of clinically relevant domains or nonsense mediated decay can constitute up to 11.9% of overlapping natural junctions, suggesting that aberrant splicing can be tolerated up to this level. Nanopore sequencing of whole BARD1 transcripts characterized 16 alternative isoforms from healthy controls, revealing that the most complex transcripts combined only two alternative splicing events. Bioinformatic analysis of ClinVar submitted variants at or near BARD1 splice sites suggest that all consensus splice site variants in BARD1 should be considered likely pathogenic, with the possible exception of variants at the donor site of exon 5. Conclusions: No BARD1 candidate rescue transcripts were identified in this study, indicating that all premature translation-termination codons variants can be annotated as PVS1. Furthermore, our analysis suggests that all donor and acceptor (IVS+/-1,2) variants can be considered PVS1 or PVS1_strong, with the exception of variants targeting the exon 5 donor site, that we recommend considering as PVS1_moderate.
| S-EPMC6877745 | biostudies-literature
Project description:ObjectiveTo determine whether the COVID-19 mRNA vaccines can negatively impact the semen parameters of young healthy men in the long-term.DesignWe conducted semen analyses on 12 men before, 3 and 9 months after achieving fully vaccinated status. Individuals who admitted a history of infertility or previous azoospermia were excluded from study participation.SubjectsHealthy male volunteers between the ages of 18-50 years old were recruited between September 2021 - March 2022.Main outcome measuresSemen analyses were performed and evaluated volume, sperm concentration, total motility, and total motile sperm count (TMSC). The primary outcome was median change in the TMSC at baseline, 3 months, and at least 9 months following vaccination.ResultsA total of 12 men volunteered in our study (median age 26 [25 - 30] years). Subjects provided follow-up semen samples at a median of 10 months following the second vaccine dose. There were no significant changes in any semen parameters between baseline, 3 months, and 10 months following vaccination. Baseline samples demonstrated median sperm concentrations and TMSC of 29.5 million/cc [9.3 - 49] and 31 million [4-51.3], respectively. At 9-month follow-up, sperm concentration and TMSC were 43 [20.5 - 63.5] (P=.351) and 37.5 [8.5 - 117.8] (P=.519), respectively. Of note, there were no significant changes in semen volume nor total motility (%) for participants at follow-up.ConclusionCOVID-19 mRNA vaccines and the booster dose does not appear to negatively impact the semen parameters of healthy males up to 10 months following vaccination.
| S-EPMC9345661 | biostudies-literature
Project description:1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.
| S-EPMC1198432 | biostudies-other
Project description:RFamide-related peptide (RFRP), the mammalian homolog of avian gonadotropin-inhibitory hormone, has a pronounced suppressive action on the reproductive axis across species. In mammals, RFRP acts directly on GnRH neurons, and likely at the level of the pituitary, to inhibit gonadotropin secretion. In the present study, we examined whether RFRP might act outside of mammalian brain on reproductive tissues directly. Using RT-PCR and in situ hybridization, we found that both RFRP and its receptors [G protein-coupled receptor (GPR) 147 and GPR74] are expressed in the testis of Syrian hamster. These results were confirmed and extended using double- and triple-label immunohistochemistry. RFRP expression was observed in spermatocytes and in round to early elongated spermatids. Significant expression of RFRP was not seen in Leydig cells. GPR147 protein was observed in myoid cells in all stages of spermatogenesis, pachytene spermatocytes, maturation division spermatocytes, and in round and late elongated spermatids. GPR74 proteins only appeared in late elongated spermatids. Additionally, we found that RFRP and its receptor mRNA are markedly altered by day length and reproductive condition. These findings highlight a possible novel autocrine and/or paracrine role for RFRP in Syrian hamster testis, potentially contributing to the differentiation of spermatids during spermiogenesis.
| S-EPMC2817627 | biostudies-literature
Project description:Tpa1 (for termination and polyadenylation) from Saccharomyces cerevisiae is a component of a messenger ribonucleoprotein (mRNP) complex at the 3' untranslated region of mRNAs. It comprises an N-terminal Fe(II)- and 2-oxoglutarate (2OG) dependent dioxygenase domain and a C-terminal domain. The N-terminal dioxygenase domain of a homologous Ofd1 protein from Schizosaccharomyces pombe was proposed to serve as an oxygen sensor that regulates the activity of the C-terminal degradation domain. Members of the Tpa1 family are also present in higher eukaryotes including humans. Here we report the crystal structure of S. cerevisiae Tpa1 as a representative member of the Tpa1 family. Structures have been determined as a binary complex with Fe(III) and as a ternary complex with Fe(III) and 2OG. The structures reveal that both domains of Tpa1 have the double-stranded beta-helix fold and are similar to prolyl 4-hydroxylases. However, the binding of Fe(III) and 2OG is observed in the N-terminal domain only. We also show that Tpa1 binds to poly(rA), suggesting its direct interaction with mRNA in the mRNP complex. The structural and functional data reported in this study support a role of the Tpa1 family as a hydroxylase in the mRNP complex and as an oxygen sensor.
| S-EPMC2847220 | biostudies-literature
Project description:(1) Background: The degradation products of ribonucleic acid (RNA)are widely used in the food and pharmaceutical industry for their flavoring and nutritional enhancement functions. Yeast is the main source for commercial RNA production, and an efficient strain is the key to reducing production costs; (2) Methods: A mutant Saccharomyces pastorianus G03H8 with a high RNA yield was developed via ARTP mutagenesis and fed-batch fermentation was applied to optimize production capacity. Genome sequencing analysis was used to reveal the underlying mechanism of higher RNA production genetic differences in the preferred mutant; (3) Results: Compared with the highest RNA content of the mutant strain, G03H8 increased by 40% compared with the parental strain G03 after response surface model optimization. Meanwhile, in fed-batch fermentation, G03H8's dry cell weight (DCW) reached 60.58 g/L in 5 L fermenter by molasses flowing and RNA production reached up to 3.58 g/L. Genome sequencing showed that the ribosome biogenesis, yeast meiosis, RNA transport, and longevity regulating pathway were closely related to the metabolism of high RNA production; (4) Conclusion: S. pastorianus G03H8 was developed for RNA production and had the potential to greatly reduce the cost of RNA production and shorten the fermentation cycle. This work lays the foundation for efficient RNA content using S. pastorianus.
| S-EPMC9497889 | biostudies-literature
Project description:In rats fed on a protein-deficient diet, albumin synthesis as a percentage of total liver protein synthesis falls from the normal value of approx. 15% to about 8%. We have extracted total cytoplasmic RNA from individual rat livers and measured the concentration of active albumin mRNA by translation in a reticulocyte lysate system from which the endogenous mRNA had been removed [Pelham & Jackson (1976) Eur. J. Biochem. 67, 247-256]. In this messenger-dependent system it is possible to measure the synthesis of albumin as a proportion of the overall protein synthesis promoted by the addition of the hepatic RNA. The results show that the concentration of translatable albumin mRNA in samples of total cytoplasmic RNA from livers of protein-deficient rats is decreased markedly. These findings suggest that dietary protein supply affects selectively the synthesis and/or functional stability of albumin mRNA in rat liver.
| S-EPMC1185671 | biostudies-other
Project description:Eaf3 is a component of both NuA4 histone acetyltransferase and Rpd3S histone deacetylase complexes in Saccharomyces cerevisiae. It is involved in the regulation of the global pattern of histone acetylation that distinguishes promoters from coding regions. Eaf3 contains a chromo domain at the N terminus that can bind to methylated Lys-36 of histone H3 (H3K36). We report here the crystal structures of the Eaf3 chromo domain in two truncation forms. Unlike the typical HP1 and Polycomb chromo domains, which contain a large groove to bind the modified histone tail, the Eaf3 chromo domain assumes an autoinhibited chromo barrel domain similar to the human MRG15 chromo domain. Compared with other chromo domains, the Eaf3 chromo domain contains a unique 38-residue insertion that folds into two short beta-strands and a long flexible loop to flank the beta-barrel core. Both isothermal titration calorimetry and surface plasmon resonance studies indicate that the interaction between the Eaf3 chromo domain and the trimethylated H3K36 peptide is relatively weak, with a K(D) of approximately 10(-4) m. NMR titration studies demonstrate that the methylated H3K36 peptide is bound to the cleft formed by the C-terminal alpha-helix and the beta-barrel core. Site-directed mutagenesis study and in vitro binding assay results show that the conserved aromatic residues Tyr-23, Tyr-81, Trp-84, and Trp-88, which form a hydrophobic pocket at one end of the beta-barrel, are essential for the binding of the methylated H3K36. These results reveal the molecular mechanism of the recognition and binding of the methylated H3K36 by Eaf3 and provide new insights into the functional roles of the Eaf3 chromo domain.
| S-EPMC2662307 | biostudies-literature