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Supplemental material, sj-docx-1-npx-10.1177_1934578X231157145 for Chemical Constituents From the Marine Microalgae Thraustochytrium pachydermum by Nguyen Thi Thu Thuy, Hoang Thi Minh Hien, Nguyen Cam Ha and Le Thi Thom, Dang Diem Hong, Nguyen Van Thinh, Nguyen Trong Dan, Nguyen Dang Hoi, Vu Thi Loan, Hoang Duc Quang, Dan Thi Thuy Hang, Phan Van Kiem, Nguyen Tien Dat, Nguyen Xuan Nhiem in Natural Product Communications

Marine microalgae are an important source of raw materials for the extraction of highly bioactive substances for humans and domestic animals. In this study, the biological characteristics and the ability to grow biomass of green marine microalga of Dunaliella tertiolecta NY isolated from seawater of Namyit Island belonging to Spratly Islands, Vietnam (in May-June, 2021) was presented. Scientific name of the strain Dunaliella tertiolecta NY based on morphological characteristics and analysis of the sequence of rDNA ITS1-5.8S-ITS2 region was identified (with accession number of this gene region of this strain was supplied on the GenBank is OM 101011). At the best conditions for the growth (i.e. Walne medium, initial cell density of 2.0 x 106 cells/mL, growth temperature of 30oC, light intensity of 60 - 100 µmol/m2s, pH 7, salinity of 30-40‰), highest NY strain cell density of 8.63 x 106 cells/mL was obtained after 15 days of culture. The microalga D. tertiolecta NY was also successfully cultured on a pilot scale in the plastic bottles 10 L and closed photobioreactors 20 - 100 L resulting in a high biomass productivity of 285 mg/L/day and a biomass rich in polyunsaturated fatty acids such as gamma - linolenic acid (C18:3 ω-6; 5.07±0.59 % of total fatty acid - TFA), alpha - linolenic acid (ALA, C18:3 ω-3, 61.79±1.98 % of TFA) and stearidonic acid (SDA; C18: 4 ω-3, 1.59±0.65 % of TFA) qualified for the extraction of value bioactive compounds.

In this study, marine microalgal communities in 12 islands belonging to the sea water region of Truong Sa Islands (Spratly Islands), Vietnam in May-June and October-November 2021 was studied. There were 305 species of marine microalgae belonging to 7 phyla were identified in 12 islands in this sea water region of Truong Sa Islands (Spratly Islands) in 2021 in which with 39 dominant species, highest species number of 147 in Central (London) Reef (D1) and the lowest species number of 15 in Ambonya Cay (D4) were detected. Up to 2021, in the sea water region of the Truong Sa Islands (Spratly Islands), 14 genera of toxic harmful algae have appeared. The average value of biodiversity index (H') and the diversity value index (Dv) in all 12 studied islands in the Truong Sa Islands (Spratly Islands) in 2021 is 3.61±0.40 and 3.43±0.39, respectively, showing that the diversity of marine microalgae of this sea water region is rich and the quality of its sea water is generally good. It has been determined that two main factors NO3- and oil content, followed by DO, salinity, temperature and Hg content in seawater were affected the density of marine microalgal cells in the sea water of Truong Sa Islands (Spratly Islands) in 2021. In which, the marine microalgal cell density has a negative correlation with temperature, salinity, NO3- and Hg content of seawater and has a positive correlation with DO parameter was discovered.

The application of nanotechnology in agriculture is a promising way to increase crop production and yield, reduce the amount of fertilizer, increase the storage time of the productions and protect the environmental sustainability. Soybean is one of the four important crops of Vietnam's agriculture and a source of food for humans and domestic animals. In this study, the effect of zerovalent cobalt nanoparticles with different concentrations (0; 0.17; 0.33 and 100 mg/kg seed) on chlorophyll a content and photosynthesis parameters of soybean plant Glycine max (L). Merr. DT96 which was planted in the autumn-winter crop in Xuan Hoa, Phuc Yen, Vinh Phuc were carried out. The obtained results showed that zerovalent cobalt nanoparticles had a positive effect on the photosynthetic parameters of soybean plants. At the used concentration of 0.33 mg cobalt/kg seed, the chlorophyll a content and photosynthetic parameters of soybean were the highest and there was a statistically significant difference compared with control. The chlorophyll a content tends to increase gradually and reaches the maximum value of 1.77 ± 0.05 mg/g fresh leaves after 40 days of sowing. The parameters related to chlorophyll fluorescence such as Fo (initial fluorescence), Fm (maximal fluorescence), Fv/Fm (maximal photochemical efficiency), effective quantum yield of photosystem II (FPSII), photosynthetic electron transport rate (ETR), Pn (net photosynthetic rate) of soybean seedlings which were treated by cobalt nanoparticles were higher than the control (except at the concentration of 100 mg/kg). The soybean seeds treated by cobalt nanoparticles had increased the chlorophyll a content of leaves and photosynthetic efficiency, leading to improve productivity of soybean.

This paper empirically investigates the links between agricultural diversity and child nutritional status in rural Myanmar. The data for this analysis come from a two-round survey of households con...

Supplemental material, sj-docx-1-npx-10.1177_1934578X221113184 for Two New Sterol Sulfates from Marine Spider Conch Lambis Lambis Linnaeus, 1758 by Nguyen Trong Dan, Le Thi Giang, Cu Nguyen Dinh, Truong Ba Hai, Nguyen Dang Hoi, Vu Thi Loan, Dan Thi Thuy Hang, Nguyen Xuan Nhiem, Bui Huu Tai and Phan Van Kiem in Natural Product Communications

South Asia remains the region with the highest prevalence of undernourishment with India accounting for 255 million food insecure people. A worsening of child nutritional outcomes has been observed in many Indian states recently and children in rural areas have poorer nutrition compared to those in urban areas. This paper investigates the relationship between land ownership, non-farm livelihoods, food security, and child nutrition in rural India, using the Young Lives Survey. The survey covers the same rural households and children over the period 2002–2013 in the states of Andhra Pradesh and Telangana. Our empirical analysis uses a sample of 1209 children (and their households) who were aged around 1 year in 2002. Our results show that large agricultural land ownership is significantly associated with better child nutrition (measured using height-for-age and stunting) and household food security. A transition from farm to non-farm work improves child nutrition, but only among landless households. While access to land is still critical for improving household food and nutrition security among rural households, there is a trend towards greater non-farm livelihoods, and a decline in reliance on farming, particularly among landless and marginal farmers.

Research on the association between armed conflict and son preference has largely been based on single-country studies, often presenting descriptive patterns. This paper empirically analyzes the association between conflict and son preference using a sample of more than 1.1 million individuals from 58 countries over the period 2003-2018. We empirically show that both the incidence and intensity of conflict exposure are associated with greater son preference. Moreover, conflict-exposed individuals are likely to realise their preference for sons, as reflected in the systematically higher prevalence of sons over daughters among these individuals. To explore the aggregate effects of these findings, we conduct a cross-country analysis of sex ratios and show that history of conflict exposure plays an important role in explaining the cross-country differences in sex ratios.

Background Violent conflicts are observed in many parts of the world and have profound impacts on the lives of exposed individuals. The limited evidence available from specific country or region contexts suggest that conflict exposure may reduce health service utilization and have adverse affects on health. This study focused on identifying the association between conflict exposure and continuum of care (CoC) services that are crucial for achieving improvements in reproductive, maternal, newborn, and child health and nutrition (RMNCHN). Methods and findings We combined data from 2 sources, the Demographic Health Surveys (DHS) and the Uppsala Conflict Data Program’s (UCDP) Georeferenced Event Dataset, for a sample of 452,192 women across 49 countries observed over the period 1997 to 2018. We utilized 2 consistent measures of conflict—incidence and intensity—and analyzed their association with maternal CoC in 4 key components: (i) at least 1 antenatal care (ANC) visit; (ii) 4 or more ANC visits; (iii) 4 or more ANC visits and institutional delivery; and (iv) 4 or more ANC visits, institutional delivery, and receipt of postnatal care (PNC) either for the mother or the child within 48 hours after birth. To identify the association between conflict exposure and components of CoC, we estimated binary logistic regressions, controlling for a large set of individual and household-level characteristics and year-of-survey and country/province fixed-effects. This empirical setup allows us to draw comparisons among observationally similar women residing in the same locality, thereby mitigating the concerns over unobserved heterogeneity. Around 39.6% (95% CI: 39.5% to 39.7%) of the sample was exposed to some form of violent conflict at the time of their pregnancy during the study period (2003 to 2018). Although access to services decreased for each additional component of CoC in maternal healthcare for all women, the dropout rate was significantly higher among women who have been exposed to conflict, relative to those who have not had such exposure. From logistic regression estimates, we observed that relative to those without exposure to conflict, the odds of utilization of each of the components of CoC was lower among those women who were exposed to at least 1 violent conflict. We estimated odds ratios of 0.86 (95% CI: 0.82 to 0.91, p < 0.001) for at least 1 ANC; 0.95 (95% CI: 0.91 to 0.98, p < 0.005) for 4 or more ANC; and 0.92 (95% CI: 0.89 to 0.96, p < 0.001) for 4 or more ANC and institutional delivery. We showed that both the incidence of exposure to conflict as well as its intensity have profound negative implications for CoC. Study limitations include the following: (1) We could not extend the CoC scale beyond PNC due to inconsistent definitions and the lack of availability of data for all 49 countries across time. (2) The measure of conflict intensity used in this study is based on the number of deaths due to the absence of information on other types of conflict-related harms. Conclusions This study showed that conflict exposure is statistically significantly and negatively associated with utilization of maternal CoC services, in each component of the CoC scale. These findings have highlighted the challenges in achieving the Sustainable Development Goal 3 in conflict settings, and the need for more concerted efforts in ensuring CoC, to mitigate its negative implications on maternal and child health., Anu Rammohan and co-workers study associations between exposure to violent conflicts and take-up of maternal health care services across countries., Author summary Why was this study done? The concept of continuum of care (CoC) has emerged as an important guiding principle targeting improvements in reproductive, maternal, newborn, and child health and nutrition (RMNCHN). Previous literature on the links between conflicts and CoC has examined the role of socioeconomic and demographic factors on CoC and focused on single country settings at one point in time. The influence of conflict exposure on the uptake of CoC services has not been previously analyzed in a multicountry context, and little is known on the links between conflict exposure and CoC utilization using consistent measures of conflict and CoC for a large group of countries over a period of time. What did the researchers do and find? We combined georeferenced data from 2 major sources for a sample of 452,192 women across 49 countries over the period 1997 to 2018. We estimated binary logistic regression models on the link between conflict exposure and 4 components of CoC in maternal health services, controlling for a wide range of individual- and household-level characteristics as well as year-of-survey and country/province fixed effects. Exposure to conflict—both in terms of incidence and intensity—was found to be negatively associated with CoC across the entire spectrum. What do these findings mean? This study showed that violent conflict is an important driver of disruptions in CoC in developing countries. More policy efforts need to be directed to ensuring CoC in maternal and child health services in conflict-prone parts of the world.

This paper aims to investigate the classification power of market variables as predictors in the financial distress prediction model for listed companies in a frontier market as Vietnam securities market. Data is collected from 70 financially distressed companies that suffer a loss in 3 consecutive years and 156 non-financially distressed companies in Vietnam from 2010 to 2017. Four different models have been constructed using Logit regression and SVM analysis technique to make a prediction in 1 to 3-year ahead. The analysis results show that combining accounting ratios with market variables such as price volatility and P/E can improve the classification ability of the ex-ante model. In addition, contrary to the results of related previous researches in emerging markets, in this study, Logit models outperform SVM models. Therefore, for future research, models that apply other machine learning classifiers such as Decision Tree (DT) or Neural Network (NN) should be investigated., Este artículo tiene como objetivo investigar el poder de clasificación de las variables del mercado como factores predictivos en el modelo de predicción de dificultades financieras para las empresas que cotizan en bolsa en un mercado fronterizo como el mercado de valores de Vietnam. Los datos se recopilan de 70 compañías con dificultades financieras que sufrieron una pérdida en 3 años consecutivos y 156 empresas sin dificultades financieras en Vietnam desde 2010 a 2017. Se han construido cuatro modelos diferentes utilizando regresión Logit y la técnica de análisis de SVM para hacer una predicción en 1 a 3 años por delante. Los resultados del análisis muestran que la combinación de ratios contables con variables de mercado como la volatilidad de los precios y el P / E puede mejorar la capacidad de clasificación del modelo ex ante. Además, a diferencia de los resultados de investigaciones anteriores relacionadas en mercados emergentes, en este estudio, los modelos Logit superan a los modelos SVM. Por lo tanto, para futuras investigaciones, se deben investigar los modelos que aplican otros clasificadores de aprendizaje automático, como el Árbol de decisiones (DT) o la Red neuronal (NN).

Ecdysteroids are steroidal hormones that have been originally found in the animal kingdom to control moulting (ecdysis) and other metamorphosic processes in insects. Structurally, they are polyhydroxylated compounds that have a four-ringed skeleton, usually composed of 27, 28 or 29 carbon atoms. This group of naturally occurring plant ecdysteroids was designated phytoecdysteroids (Pes) to differentiate them from those isolated from animal sources. Recently more and more researchs have focused, on the effects of Pes on vertebrates and humans. The range of effects of these compounds appears to be very wide. They are apparently nontoxic to mammals, most of them are useful for humans. They have a broad spectrum of pharmacological and medicinal properties in mammals, including hepatoprotective and hypoglycaemic effects, increase adaptation and anabolic effects, without androgenic side-effects. Pes are also being considered as nutraceutical additives to food products.

Phytoecdysteroids are secondary metabolites which were found in many plant species. In mammals, including humans, phytoecdysteroids don’t have hormonal activity, low toxicity and are proven to have many pharmacological effects. 20-hydroxyecdysone (20E) is one of the most common phytoecdysteroids which was detected in plants. In this study, 20E was detected in some plants of Vitex and Sida genera of the Nam Cat Tien national park. HPLC method was used for determining 20E. 3,44% was the highest concentration of 20E picked out from young Vitex tripinnata leaves. Vitex canescens and Vitex tripinnata were considered as potential plants for producing ecdysteroid-containing drugs.

Beta hemolytic streptococci group A, C and G are bacteria that cause pharyngitis, lymphadenitis, pyoderma, erysipelas, toxic shock syndrome, etc which are commonly appeared in children of different ages. In order to study the epidemiological and genetical characteristics of β hemolytic steptococci in primary school pupils in some areas of Vietnam, a total of 1450 primary school pupils including 734 males (50.62%) and 716 females (49.38%) representing for six provinces of Vietnam was investigated. The results showed that the beta hemolytic streptococci infection rate in primary school pupils was 9.38%, in which group A, group C, and group G were 3.45%, 0.55%, and 5.38%, respectively. The highest and lowest rate of streptococcal infection rate was in Quang Tri (25.5%) and Tay Ninh (4.67%) respectively. Chronic pharyngitis and tonsillitis contributed to the increment of streptococcal infection. The 11 emm types of S. pyogenes strains were identified and it hypothesized that the different existence were caused by the diversity of genotypes in various geographical regions. Genetical diversity of S. anginosus strains has been detected based on toxic genes.

Antibiotic resistance is a global problem, and serious threat to human health. Study on antibiotic susceptibility in streptococci strains group A, C, G isolated from primary school pupils in Viet Nam during 2012÷2015 showed that all streptococci strains were susceptible to cefalosporins and vancomycin as well, simultaneous resistance to amikacin. The resistant rate of GAS to tetracycline and doxycillin were 70% and 75%, respectively. The rate of erythromycin tolerance of GAS was 52.5% and 40.74% in that of GCS/GGS. The third and fourth generation cephalosporin antibiotic group and vancomycin are considered as essential drugs for treating group A, C and G streptococcal infections. The correlation between mefA and ermTR genes in streptococci genome in the macrolide resistant mechanism were not detected. A tetracycline resistant gene in the jumping gene of the GAS genome was found.

The synthesis of ribosomes in eukaryotic cells is a complex process involving many nonribosomal protein factors and snoRNAs. In general, the processes of rRNA transcription and ribosome assembly are treated as temporally or spatially distinct. Here, we describe the identification of a point mutation in the second largest subunit of RNA polymerase I near the active center of the enzyme that results in an elongation-defective enzyme in the yeast Saccharomyces cerevisiae. In vivo, this mutant shows significant defects in rRNA processing and ribosome assembly. Taken together, these data suggest that transcription of rRNA by RNA polymerase I is linked to rRNA processing and maturation. Thus, RNA polymerase I, elongation factors, and rRNA sequence elements appear to function together to optimize transcription elongation, coordinating cotranscriptional interactions of many factors/snoRNAs with pre-rRNA for correct rRNA processing and ribosome assembly.

The 35S rRNA genes at the RDN1 locus in Saccharomyces cerevisiae can be transcribed by RNA polymerase (Pol) II in addition to Pol I, but Pol II transcription is usually silenced. The deletion of RRN9 encoding an essential subunit of the Pol I transcription factor, upstream activation factor, is known to abolish Pol I transcription and derepress Pol II transcription of rRNA genes, giving rise to polymerase switched (PSW) variants. We found that deletion of histone deacetylase gene RPD3 inhibits the appearance of PSW variants in rrn9 deletion mutants. This inhibition can be explained by the observed specific inhibition of Pol II transcription of rRNA genes by the rpd3Delta mutation. We propose that Rpd3 plays a role in the maintenance of an rRNA gene chromatin structure(s) that allows Pol II transcription of rRNA genes, which may explain the apparently paradoxical previous observation that rpd3 mutations increase, rather than decrease, silencing of reporter Pol II genes inserted in rRNA genes. We have additionally demonstrated that Rpd3 is not required for inhibition of Pol I transcription by rapamycin, supporting the model that Tor-dependent repression of the active form of rRNA genes during entry into stationary phase is Rpd3 independent.

Nucleosomes and their histone components have generally been recognized to act negatively on transcription. However, purified upstream activating factor (UAF), a transcription initiation factor required for RNA polymerase (Pol) I transcription in Saccharomyces cerevisiae , contains histones H3 and H4 and four nonhistone protein subunits. Other studies have shown that histones H3 and H4 are associated with actively transcribed rRNA genes. To examine their functional role in Pol I transcription, we constructed yeast strains in which synthesis of H3 is achieved from the glucose-repressible GAL10 promoter. We found that partial depletion of H3 (≈50% depletion) resulted in a strong inhibition (>80%) of Pol I transcription. A combination of biochemical analysis and electron microscopic (EM) analysis of Miller chromatin spreads indicated that initiation and elongation steps and rRNA processing were compromised upon histone depletion. A clear decrease in relative amounts of UAF, presumably caused by reduced stability, was also observed under the conditions of H3 depletion. Therefore, the observed inhibition of initiation can be explained, in part, by the decrease in UAF concentration. In addition, the EM results suggested that the defects in rRNA transcript elongation and processing may be a result of loss of histones from rRNA genes rather than (or in addition to) an indirect consequence of effects of histone depletion on expression of other genes. Thus, these results show functional importance of histones associated with actively transcribed rRNA genes.

It is known that mutations in gene SIR2 increase and those in FOB1 decrease recombination within rDNA repeats as assayed by marker loss or extrachromosomal rDNA circle formation. SIR2-dependent chromatin structures have been thought to inhibit access and/or function of recombination machinery in rDNA. We measured the frequency of FOB1-dependent arrest of replication forks, consequent DNA double-strand breaks, and formation of DNA molecules with Holliday junction structures, and found no significant difference between sir2Δ and SIR2 strains. Formal genetic experiments measuring mitotic recombination rates within individual rRNA genes also showed no significant difference between these two strains. Instead, we found a significant decrease in the association of cohesin subunit Mcd1p (Scc1p) to rDNA in sir2Δ relative to SIR2 strains. From these and other experiments, we conclude that SIR2 prevents unequal sister-chromatid recombination, probably by forming special cohesin structures, without significant effects on recombinational events within individual rRNA genes.

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.

About half of approximately 150 rRNA genes are transcriptionally active in Saccharomyces cerevisiae. Chromatin structures in the inactive, and not the active, copies were previously thought to silence both rRNA genes and reporter Pol II genes. Contrary to this belief, we found that silencing of reporters is much stronger in a mutant with approximately 25 rDNA copies, all of which are transcriptionally active. By integrating reporter gene mURA3 with an inactive rDNA copy missing the Pol I promoter, we found that mURA3 is not silenced in chromosomal rDNA repeats. Together with the demonstration of a requirement for active Pol I in silencing, these results show a reciprocal relationship in gene expression between Pol I and Pol II in rDNA.

At the end of the 35S rRNA gene within ribosomal DNA (rDNA) repeats in Saccharomyces cerevisiae lies an enhancer that has been shown to greatly stimulate rDNA transcription in ectopic reporter systems. We found, however, that the enhancer is not necessary for normal levels of rRNA synthesis from chromosomal rDNA or for cell growth. Yeast strains which have the entire enhancer from rDNA deleted did not show any defects in growth or rRNA synthesis. We found that the stimulatory activity of the enhancer for ectopic reporters is not observed in cells with disrupted nucleolar structures, suggesting that reporter genes are in general poorly accessible to RNA polymerase I (Pol I) machinery in the nucleolus and that the enhancer improves accessibility. We also found that a fob1 mutation abolishes transcription from the enhancer-dependent rDNA promoter integrated at the HIS4 locus without any effect on transcription from chromosomal rDNA. FOB1 is required for recombination hot spot (HOT1) activity, which also requires the enhancer region, and for recombination within rDNA repeats. We suggest that Fob1 protein stimulates interactions between rDNA repeats through the enhancer region, thus helping ectopic rDNA promoters to recruit the Pol I machinery normally present in the nucleolus.

Initiation of ribosomal DNA (rDNA) transcription by RNA polymerase I (Pol I) in the yeast Saccharomyces cerevisiae involves upstream activation factor (UAF), core factor, the TATA binding protein (TBP), and Rrn3p in addition to Pol I. We found previously that yeast strains carrying deletions in the UAF component RRN9 switch completely to the use of Pol II for rRNA transcription, with no residual Pol I transcription. These polymerase-switched strains initially grow very slowly, but subsequent expansion in the number of rDNA repeats on chromosome XII leads to better growth. Recently, it was reported that TBP overexpression could bypass the requirement of UAF for Pol I transcription in vivo, producing nearly wild-type levels of growth in UAF mutant strains (P. Aprikian, B. Moorefield, and R. H. Reeder, Mol. Cell. Biol. 20:5269-5275, 2000). Here, we demonstrate that deletions in the UAF component RRN5, RRN9, or RRN10 lead to Pol II transcription of rDNA. TBP overexpression does not suppress UAF mutation, and these strains continue to use Pol II for rRNA transcription. We do not find evidence for even low levels of Pol I transcription in UAF mutant strains carrying overexpressed TBP. In diploid strains lacking both copies of the UAF component RRN9, Pol II transcription of rDNA is more strongly repressed than in haploid strains but TBP overexpression still fails to activate Pol I. These results emphasize that UAF plays an essential role in activation of Pol I transcription and silencing of Pol II transcription of rDNA and that TBP functions to recruit the Pol I machinery in a manner completely dependent on UAF.

Strains of the yeast Saccharomyces cerevisiae defective in transcription factor UAF give rise to variants able to grow by transcribing endogenous ribosomal DNA (rDNA) by RNA polymerase II (Pol II). We have demonstrated that the switch to growth using the Pol II system consists of two steps: a mutational alteration in UAF and an expansion of chromosomal rDNA repeats. The first step, a single mutation in UAF, is sufficient to allow Pol II transcription of rDNA. In contrast to UAF mutations, mutations in Pol I or other Pol I transcription factors can not independently lead to Pol II transcription of rDNA, suggesting a specific role of UAF in preventing polymerase switch. The second step, expansion of chromosomal rDNA repeats to levels severalfold higher than the wild type, is required for efficient cell growth. Mutations in genes that affect recombination within the rDNA repeats, fob1 and sir2, decrease and increase, respectively, the frequency of switching to growth using Pol II, indicating that increased rDNA copy number is a cause rather than a consequence of the switch. Finally, we show that the switch to the Pol II system is accompanied by a striking alteration in the localization and morphology of the nucleolus. The altered state that uses Pol II for rDNA transcription is semistable and heritable through mitosis and meiosis. We discuss the significance of these observations in relation to the plasticity of rDNA tandem repeats and nucleolar structures as well as evolution of the Pol I machinery.

Srp1p, the protein encoded by SRP1 of the yeast Saccharomyces cerevisiae, is a yeast nuclear localization signal (NLS) receptor protein. We have previously reported isolation of a protein kinase from yeast extracts that phosphorylates Srp1p complexed with NLS peptides/proteins. From partial amino acid sequences of the four subunits of the purified kinase, we have now identified this protein kinase to be identical to yeast casein kinase II (CKII). It was previously thought that autophosphorylation of the 36 kDa subunit of the yeast enzyme was stimulated by the substrate, GST-Srp1p. However, with the use of a more refined system, no stimulation of autophosphorylation of the 36 kDa subunit of yeast CKII was observed. Biochemical and mutational analyses localized the in vitro phosphorylation site of Srp1p by CKII to serine 67. It was shown that, in the absence of NLS peptides/proteins, phosphorylation of the intact Srp1p protein is very weak, but deletion of the C-terminal end causes great stimulation of phosphorylation without NLS peptides/proteins. Thus, the CKII phosphorylation site is apparently masked in the intact protein structure by the presence of a C-terminal region, probably between amino acids 403 and 516. Binding of NLS peptides/proteins most likely causes a change in protein conformation, exposing the CKII phosphorylation site. Mutational alterations of serine 67, the CKII phosphorylation site, to valine (S67V) and aspartic acid (S67D) were not found to cause any significant deleterious effects on cell growth. Analysis of in vivo phosphorylation showed that at least 30% of the wild type Srp1p molecules are phosphorylated in growing cells, and that the phosphorylation is mostly at the serine 67 CKII site. The ability of Srp1p purified from E coli and treated with calf intestinal phosphatase to bind a SV40 T-antigen NLS peptide was compared with that of Srp1p which was almost fully phosphorylated by CKII. No significant difference was observed. It appears that NLS binding does not require any phosphorylation of Srp1p, either by CKII or by some other protein kinase.

Srp1p, the protein encoded by SRP1 of Saccharomyces cerevisiae, is a nuclear-pore-associated protein. Its Xenopus homolog, importin, was recently shown to be an essential component required for nuclear localization signal (NLS)-dependent binding of karyophilic proteins to the nuclear envelope [Gorlich, D., Prehn, S., Laskey, R. A. & Hartman, E. (1994) Cell 79, 767-778]. We have discovered a protein kinase whose activity is stimulated by Srp1p (Srp1p fused to glutathione S-transferase and expressed in Escherichia coli) and is detected by phosphorylation of Srp1p and of a 36-kDa protein, a component of the protein kinase complex. The enzyme, called Srp1p kinase, is a protein-serine kinase and was found in extracts in two related complexes of approximately 180 kDa and 220 kDa. The second complex, when purified, contained four protein components including the 36-kDa protein. We observed that, upon purification of the kinase, phosphorylation of Srp1p became very weak, while activation of phosphorylation of the 36-kDa protein by Srp1p remained unaltered. Significantly, NLS peptides and the nuclear proteins we have tested greatly stimulated phosphorylation of Srp1p, suggesting that Srp1p, complexed with karyophilic proteins carrying an NLS, is the in vivo substrate of this protein kinase.

Previously, we have isolated mutants of Saccharomyces cerevisiae primarily defective in the transcription of 35S rRNA genes by RNA polymerase I and have identified a number of genes (RRN genes) involved in this process. We have now cloned the RRN6 and RRN7 genes, determined their nucleotide sequences, and found that they encode proteins of calculated molecular weights of 102,000 and 60,300, respectively. Extracts prepared from rrn6 and rrn7 mutants were defective in in vitro transcription of rDNA templates. We used extracts from strains containing epitope-tagged wild-type Rrn6 or Rrn7 proteins to purify protein components that could complement these mutant extracts. By use of immunoaffinity purification combined with biochemical fractionation, we obtained a highly purified preparation (Rrn6/7 complex), which consisted of Rrn6p, Rrn7p, and another protein with an apparent molecular weight of 66,000, but which did not contain the TATA-binding protein (TBP). This complex complemented both rrn6 and rrn7 mutant extracts. Template commitment experiments carried out with this purified Rrn6/7 complex and with rrn6 mutant extracts have demonstrated that the Rrn6/7 complex does not bind stably to the rDNA template by itself, but its binding is dependent on the initial binding of some other factor(s) and that the Rrn6/7 complex is required for the formation of a transcription-competent preinitiation complex. These observations are discussed in comparison to in vitro rDNA transcription systems from higher eukaryotes.