S1 Treatment Research Articles

Effect of Sulfur on the Yield and Essential and Nonessential Element Composition of Alfalfa Determined by Polarized Energy Dispersive X‐ray Fluorescence

Alfalfa crops were grown in the field at the University of Ankara (473939 E, 4385149 N), over two seasons between 2001 and 2003 with sulfur (S) supplied at two different rates. The experimental design was a randomized complete block with five replicates and three cutting dates in each season. Sulfur was applied at rates of 0 (control), 160 (S1), and 240 (S2) kg ha−1 as gypsum. Alfalfa hay was harvested three times each year, and the concentrations of elements in the hay were measured by polarized energy dispersive x‐ray fluorescence (PEDXRF). Sulfur fertilization increased S concentrations and improved alfalfa hay yield for both years. Applied S slightly reduced phosphorus (P) and magnesium (Mg) concentrations in the first year and had no significant effect on the potassium (K) and calcium (Ca) concentrations of alfalfa hay for either year. The highest S (240 kg ha−1 S) level increased sodium (Na) concentrations in the first year but decreased them in the second year. Sulfur application also decreased chloride (Cl) concentration in the first year. Molybdenum (Mo) concentration of the alfalfa was significantly reduced by S1 treatment in year 1. Iron (Fe) concentration was increased by S2 treatment in the second year, and zinc (Zn) concentration was increased by S1 treatment in the first year. However, applied S had no effect on manganese (Mn), copper (Cu), titanium (Ti), vanadium (V), cobalt (Co), nickel (Ni), barium (Ba), uranium (U), and lead (Pb) concentrations for either year. Applied S decreased aluminum (Al) and silicon (Si) and increased bromine (Br) and rubidium (Rb) concentrations of alfalfa in the first year. In addition, strontium (Sr) concentration was increased by S2 treatment in the first year but was decreased in the second year. The stage of cutting greatly affected mineral concentrations. Compared with the first cutting, S concentrations were higher in the second and third harvest for both years. In general, the concentrations of P, Mg, Na, Cl, Mo, Ti, V, Br, Co, Ba, Sr, Rb, U, and Pb were increased, whereas the concentrations of Fe, Zn, Mn, Cu, and Ni were decreased with later cutting. The concentrations of K and Ca did not vary between cuts.

호맥 사일리지의 급여가 비육돈의 생산성, 혈액성상 및 도체특성에 미치는 효과

본 연구는 조사료인 호맥 사일리지를 배합사료와 혼합급여 시 비육돈의 성장, 혈액성상 및 도체특성에 미치는 영향을 알아보기 위해 시험을 실시하였다. 3원 교잡종(<TEX>$Landrace{\times}Yorkshire{\times}Duroc$</TEX>) 비육돈 18두를 공시하였으며 시험개시시 평균체중은 <TEX>$74.22{\pm}0.71kg$</TEX>이었고 28일간 개체별 사양시험을 실시하였다. 시험설계는 호맥 사일리지 첨가에 따라 1) Con(basal diet), 2) S1(basal diet+1.66% rye silage), 3) S2(basal diet+3.32% rye silage)로 3처리로 하여 처리당 6반복, 반복당 1마리씩 <TEX>$1.8{\times}1.8m$</TEX> 크기의 돈방에 개별 수용하였다. 생산성에서 <TEX>$4{\sim}7$</TEX>주 동안 일당사료 섭취량에서는 CON구가 S1구와 S2구와 비교하여 유의적으로 높게 나타났다(p<0.05). 전체 사양시험기간 동안(<TEX>$0{\sim}7$</TEX>주) 일당사료섭취량에서는 CON구와 S2구가 S1구와 비교하여 유의적으로 높게 나타났다(p<0.05). 혈중 스트레스 관련 호르몬인 코티졸 함량의 변화는 대조구와 비교하여 호맥 사일리지를 급여한 S1구와 S2구에서 유의적으로 감소하였다(p<0.05). 육색에서 명도를 나타내는 <TEX>$L^*$</TEX>값은 S2구가 CON구와 비교하여 유의적으로 높게 나타났고(p<0.05) 황색도를 나타내는 <TEX>$b^*$</TEX>값은 S1구와 S2구가 CON구와 비교하여 높게 나타났다(p<0.05). 도체 등지방 두께는 CON구가 S1구와 S2구와 비교하여 유의적으로 높게 증가하였다(p<0.05). 살코기 내 지방산 조성에서는 포화지방산 중 palmitic acid와 stearic acid의 함량은 CON구가 S1구와 S2구와 비교하여 유의적으로 높은 함량을 나타내었다(p<0.05). 불포화 지방산에서는 eicosenoic acid와 linolenic acid의 함량이 S2구가 CON구와 S1구와 비교하여 유의적으로 높았다(p<0.05). 총 포화지방산 함량은 CON구가 다른 처리구와 비교하여 가장 높았으며(p<0.05) 포화지방산과 불포화지방산 비율에서는 S1구와 S2구가 CON구와 비교하여 유의적으로 높은 결과를 보였다(p<0.05). 지방 내 지방산 조성에서는 불포화지방산 중 linolenic acid의 함량은 S2구가 S1구와 CON구와 비교하여 유의적으로 높았다(p<0.05). 건물 소화율에서는 S1구와 S2구가 CON구와 비교하여 유의적으로 높게 나타났고(p<0.05) 질소 소화율에서는 S1구가 처리구중에서 가장 높은 결과를 보였다(p<0.05). 분변 내 휘발성 지방산 발생량에서는 분을 1일간 발효 후 측정한 결과 S1구와 S2구가 CON구와 비교하여 유의적으로 높게 나타났다(p<0.05). 결론적으로, 비육돈 사료 내 3.32%의 호맥 사일리지의 혼합급여는 혈액 내 코티졸 함량, 도체육의 명도와 황색도, 지방산 조성 및 영양소 소화율에 영향을 미치는 것으로 사료되나 이에 대한 보다 많은 연구가 필요할 것으로 판단된다. This study was conducted to evaluate the effects of feeding rye silage on growth performance, blood and carcass characteristics in finishing pigs. The total of eighteen(<TEX>$Landrace{\times}Yorkshire{\times}Duroc$</TEX>) pigs(<TEX>$74.22{\pm}0.71kg$</TEX>) were used for 49-day assay. Dietary treatments included 1) CON(basal diet), 2) S1(1.66% rye silage) and 3) S2(3.32% rye silage). In growth performance, from 4 to 7 weeks, average daily feed intake(ADFI) in pigs fed CON diet was higher than pigs fed S1 and S2 diets. Through the entire experimental period(<TEX>$0{\sim}7$</TEX> weeks), a ADFI in CON and S2 treatments was higher than S1 treatment (p<0.05). Serum cortisol concentration of pigs fed rye silage decreased significantly compared to pigs fed basal diet (p<0.05). The Hunter's <TEX>$L^*$</TEX>(lightness) value of loin in the pigs fed S2 diet was higher than that of loin in the pigs fed CON diet(p<0.05). The <TEX>$b^*$</TEX>(yellowness) value of loin in the pigs fed S1 and S2 diets were higher than CON treatment(p<0.05). Backfat thickness in CON treatment increased significantly compared to S1 and S2 treatments(p<0.05). Among fatty acid contents of lean meat, the contents of palmitic and stearic acids were significantly higher in CON than others(p<0.05), where as eicosenoic and linolenic acids were the highest in S2 treatment among treatments(p<0.05). Total SFA(saturated fatty acid) was the highest in CON(p<0.05) and S1 and S2 treatments were higher USFA/SFA(unsaturated fatty acid/saturated fatty acid) ratio than CON treatment. In fats, linolenic acid was higher in S2 treatment than those of S1 and CON treatments(p<0.05). Digestibility of dry matter in pigs fed S1 and S2 diets was greater than that of pigs fed CON diet(p<0.05) and S1 treatment was higher than others on digestibility of nitrogen(p<0.05). VFA(volatile fatty acid) emission from focal, measured after 1 day, increased significantly in pigs fed S1 and S2 diets(p<0.05). In conclusions, our results showed that feeding rye silage might be beneficial in decrease of cortisol concentration, hunter's <TEX>$L^*$</TEX>(lightness) value and <TEX>$b^*$</TEX>(yellowness), fatty acid contents, and nutrient digestibility. However, there should be more study on the relationship between feeding rye silagr and finishing pigs.

Water use and lint yield response of drip irrigated cotton to the length of irrigation season

A 2-year experiment was conducted at Tal Amara Research Station in the Bekaa Valley of Lebanon to determine water use and lint yield response to the length of irrigation season of drip irrigated cotton ( Gossypium hirsutum L.). Crop evapotranspiration (ET crop) and reference evapotranspiration (ET rye-grass) were directly measured at weekly basis during the 2001 growing period using crop and rye-grass drainage lysimeters. Crop coefficients ( K c) in the different growth stages were calculated as ET crop/ET rye-grass. Then, the calculated K c values were used in the 2002 growing period to estimate evapotranspiration of cotton using the FAO method by multiplying the calculated K c values by ET rye-grass measured in 2002. The length of irrigation season was determined by terminating irrigation permanently at first open boll (S1), at early boll loading (S2), and at mid boll loading (S3). The three treatments were compared to a well-watered control (C) throughout the growing period. Lint yield was defined as a function of components including plant height at harvest, number of bolls per plant, and percentage of opened bolls per plant. Lysimeter-measured crop evapotranspiration (ET crop) totaled 642 mm in 2001 for a total growing period of 134 days, while when estimated with the FAO method in 2002 it averaged 669 mm for a total growing period of 141 days from sowing to mature bolls. Average K c values varied from 0.58 at initial growth stages (sowing to squaring), to 1.10 at mid growth stages (first bloom to first open boll), and 0.83 at late growth stages (early boll loading to mature bolls). Results showed that cotton lint yields were reduced as irrigation amounts increased. Average across years, the S1 treatment produced the highest yield of 639 kg ha −1 from total irrigations of 549 mm, compared to the S2 and S3 treatments, which yielded 577 and 547 kg ha −1 from total irrigations of 633 and 692 mm, respectively, while the control resulted in 457 kg ha −1 of lint yield from 738 mm of irrigation water. Water use efficiency (WUE) was found to be higher in S1 treatment and averaged 1.3 kg ha −1 mm −1, followed by S2 (1.1 kg ha −1 mm −1), and S3 (1.0 kg ha −1 mm −1), while in the control WUE was 0.80 kg ha −1 mm −1. Lint yield was negatively correlated with plant height and the number of bolls per plant and positively correlated with the percentage of opened bolls. This study suggests that terminating irrigation at first open boll stage has been found to provide the highest cotton yield with maximum WUE under the semi-arid conditions of the Bekaa Valley of Lebanon.

The AUUCU Repeats Responsible for Spinocerebellar Ataxia Type 10 Form Unusual RNA Hairpins

Spinocerebellar ataxia type 10 is an autosomal dominant disorder caused by expansion of a pentameric repeat tract (ATTCT.AGAAT)(n) in intron 9 of the gene that encodes ataxin-10. We have analyzed duplex DNA containing the repeat, the individual DNA strands, and the RNA that would be generated by transcription of the repeat. Circular dichroism and UV absorbance measurements suggest that the previously reported tendency of the repeat to unpair when supercoiled is probably related simply to GC content rather than reflecting any unusual property of the duplex. DNA containing d(ATTCT)9 forms a folded structure at relatively low temperatures, whereas the antisense strand, d(AGAAT)9, does not form a structure even at 0 degrees C. In contrast r(AUUCU)9 forms a folded structure under physiologically reasonable conditions. S1 nuclease analysis reveals a single region of hypersensitivity in the middle of the repeat tract, whereas V1 digestion is consistent with a hydrogen bonded or well stacked structure. CD spectroscopy shows that the structure is unimolecular and hydrogen bonded and has a significant amount of A-form helix. NMR spectroscopy demonstrates that these hydrogen bonds comprise an equal number of A.U and U.U base pairs. Our data thus suggest that the repeat forms an unusual RNA hairpin. Thus the ability to form an RNA hairpin seems to be a common property of those Repeat Expansion Diseases that are not recessively inherited and are caused by repeats that are transcribed but not translated.

Evapotranspiration and seed yield of field grown soybean under deficit irrigation conditions

Evapotranspiration was measured for a reference crop, rye grass ( Lolium prerenne) and soybean ( Glycine max L. Merril) grown over two seasons in 2000 and 2001 at Tal Amara Research Station, Lebanon, using drainage and weighing lysimeters. Climatic data from the field weather station were recorded daily. Within the experimental plots, irrigation was withheld at full bloom, R2 stage (S1 treatment), at seed enlargement, R5 stage (S2 treatment) and at mature seeds, R7 stage (S3 treatment). Further, a control (C) was fully-irrigated throughout the growing period. Average crop evapotranspiration (ETc) as measured by the drainage lysimeters in 2000 totaled 800 mm for a total growing period of 140 days. However, when ETc was measured by the weighing lysimeter in 2001, it was 725 mm during a growing period of 138 days. Average crop coefficients ( K c) were computed for different growth stages for the two growing periods by dividing the measured crop evapotranspiration (ETc) by the corresponding measured reference evapotranspiration (ETo-rye grass). K c values ranged from 0.62 at V10 stage (10th node on the main stem beginning with the unifoliolate node) to 1.0 at pod initiation, then to 0.81 at mature pods. Growth parameters, leaf area index (LAI) and dry matter accumulation, have been shown to be sensitive to water stress caused by the deficit irrigations. However, growth parameters were found to compensate for water stress at early stages, while at seed maturity the compensation ability was decreased. Plants of the lysimeters produced average aboveground biomass and seed yield of 8.1 and 3.5 t ha −1, respectively. However, in the well-irrigated field treatment, aboveground biomass and seed yield averaged 7.3 and 3.2 t ha −1, respectively. Deficit irrigation at R2 stage reduced aboveground biomass and seed yield by 16 and 4%, respectively, while deficit irrigation at R5 stage reduced these two parameters by 6 and 28%, respectively, with comparison to the control. The significant decrease in biomass at R2 stage due to water deficit may be attributed to a pronounced reduction in the number of vegetative nodes. However, limited irrigation at this stage did not reduce significantly ( P < 0.01) neither seed number nor seed weight, while at R5 stage these two parameters were reduced by 20 and 10%, respectively, with comparison to the control. Results showed also that deficit irrigation at R7 stage (S3) was more profitable than irrigation deficit at any other crop phenology and did not cause significant reductions either in seed number or seed weight.

Responses of Artemisia annua L. to lead and salt-induced oxidative stress

The Artemisia annua L. plants were subjected separately to NaCl (0–160 mM) and lead acetate (0–500 μM) at the age of 90 days (S1 treatment) and 120 days (S2 treatment). The treated plants were studied on 100, 130 and 160 days after sowing (DAS) in S1, and on 130 and 160 DAS in S2 treatments for lipid peroxidation rate, photosynthetic rate (Pn), chlorophyll content, artemisinin concentration and artemisinin yield in leaf samples and for total biomass accumulation. The treatments enhanced lipid peroxidation at all stages of plant growth and increased the concentration and yield of artemisinin at 100 and 130 DAS in S1 and S2, respectively, while other parameters declined at all growth stages. The magnitude of changes was greater in lead-treated than in salt-treated plants. Both treatments induced oxidative stress which might have damaged the photosynthetic apparatus resulting in a loss of chlorophyll content and a decline in photosynthetic rate, biomass accumulation and artemisinin production. The increase in artemisinin content, observed during the early phase of plant growth, might be due to a sudden conversion of its precursors (e.g. artemisinic acid/dihydroartemisinic acid) to artemisinin by activated oxygen species under oxidative stress.

Sequencing and Characterization of Plasmid pUIBI-1 from Bacillus thuringiensis Serovar entomocidus LBIT-113

Plasmid pUIBI-1 from Bacillus thuringiensis svr. entomocidus was sequenced and its replication mechanism analyzed. Sequence analysis revealed that pUIBI-1 contains 4671 bp and a 32% GC content. Plasmid pUIBI-1 also includes at least seven putative open reading frames (ORFs) encoding for proteins ranging from 5 to 50 kDa. ORF-1 encodes for a putative 16-kDa Rep protein, which lacks homology with proteins of similar function. ORF2 encodes for a protein of 50 kDa and shows homology with Mob proteins of plasmids pLUB1000 from Lactobacillus hilgardii (32.2%) and pGI2 from B. thuringiensis (33.7%). Detection of single-stranded DNA (ssDNA) intermediates indicated that pUIBI-1 replicates by the rolling-circle replication mechanism, as demonstrated by S1 treatment and Southern hybridization under non-denaturing conditions.

Aminoacylation of tRNA by antisense molecule

Aminoacylation of tRNA was attempted through formation of tRNA/DNA/aa-PNA (N-aminoacylated peptide nucleic acid) ternary hybrid. A 23-mer DNA, that is complementary to a 3'-terminal of tRNA and to a 9-mer PNA carrying an amino acid unit, was designed to achieve close proximity between the amino acid and the 3'-OH group of tRNA. The aminoacylation was carried out in a buffer solution containing imidazole. The aminoacylation was detected by nuclease S1 treatment followed by HPLC and MALDI-TOF MS. This novel methodology will open a way for easy and versatile aminoacylation of nonnatural amino acids onto specific tRNAs.

Nucleotide sequence and characterization of the cryptic Bacillus thuringiensis plasmid pGI3 reveal a new family of rolling circle replicons.

The complete nucleotide sequence of plasmid pGI3 from Bacillus thuringiensis subsp. thuringiensis H1.1. was obtained. Although this 11,365-bp molecule contained at least 11 putative open reading frames (ORFs), extensive database searches did not reveal any homologous sequences with the exception of ORF6, which displayed similarity to the largest ORF of pSTK1, a 1,883-bp cryptic plasmid isolated from Bacillus stearothermophilus. Deletion analysis to determine the pGI3 minimal replicon revealed that ORF6 is the rep gene. Replication occurred via a single-stranded DNA (ssDNA) intermediate, as demonstrated by S1 treatment and Southern hybridization in nondenaturating conditions. Interestingly, however, no homology was found between the pGI3 (ORF6) and pSTK1 (ORF3) rep genes and those from other single-stranded DNA plasmids, nor was there any DNA similarity to the double-strand origins of replication characterized so far, indicating that pGI3 and pSTK1 form another, new family of ssDNA plasmids. PCR analysis revealed that the pGI3 rep gene is largely distributed among B. thuringiensis strains but can also be found in B. cereus and B. mycoides strains, albeit at a lower frequency. Finally, segregation experiments performed with B. subtilis and B. thuringiensis showed that the pGI3 derivatives, including the minimal replicon, were segregationally stable at temperatures suitable for B. thuringiensis growth (<43 degrees C).

Isolation of 8-methoxypsoralen accessible DNA domains from chromatin of intact cells

The chromatin organization of living mammalian cells was probed using 8-methoxypsoralen (MOP). In intact cells, MOP intercalates into DNA domains which are also preferentially accessible to micrococcal nuclease. After UV365 nm irradiation of MOP-treated cells, this chemical forms bifunctional adducts crosslinking the two strands of DNA. Following extraction of cellular DNA, heat denaturation and renaturation at low temperature, the fraction of crosslinked DNA is obtained following enzymatic hydrolysis of unhybridized, non-crosslinked DNA by nuclease S1 treatment. An application of this procedure in the isolation of 8-methoxypsoralen-accessible DNA domains during DNA excision repair is shown.

Structural analysis of proviral DNA in simian foamy virus (LK-3)-infected cells

Proviral DNA of the T-cell lymphotropic simian foamy virus strain LK-3 was characterized. In infected cells, multiple copies of unintegrated linear duplex viral DNA of about 13 kbp length are present. Nuclease S1 treatment of the DNA generated two fragments of 6.5 and 6.0 kbp length that were cloned in phage and plasmid vectors. The proviral DNA contains a single-stranded gap of 109 nucleotides. A DNA fragment spanning the gap was cloned after completing the double strand by DNA synthesis in vitro. At the 3' end, the gap contains a polypurine tract (PPT) similar to the putative initiation site of retroviral plus strand DNA synthesis, suggesting discontinuous DNA synthesis. Further analysis of the genome architecture revealed LTRs of 1.7 kbp length. An additional 1.7 kbp DNA fragment was detected after nuclease S1 digestion of proviral DNA and probably represents trimmed intermediates of "strong-stop" DNA.

Plurality of genital human papillomaviruses: Characterization of two new types with distinct biological properties

The genomes of two new genital human papillomavirus (HPV) types, tentatively named HPVs 39 and 42, have been cloned from biopsy specimens of penile Bowenoid papules and vulvar papillomas, respectively. Blot hybridization experiments, performed under stringent conditions ( T m-10°), have revealed no cross-hybridization between the DNAs of HPVs 39 and 42, and between these DNAs and those of other genital and cutaneous HPVs. A significant cross-hybridization has been observed between the DNA of HPV42 and that of HPV32, the latter being associated with oral focal epithelial hyperplasia. The fraction of HPV32 and HPV42 hybrid molecules resistant to nuclease S1 treatment after hybridization in liquid phase at saturation has been evaluated to 20%, supporting the view that these HPVs constitute distinct types. In addition to HPV42 DNA, a 6.8-kb BamHI fragment, cross-hybridizing with HPV39 DNA, has been cloned from the vulvar papilloma DNA preparation. The cross-hybridization has been evaluated to 16%, pointing to the existence of an additional HPV39-related type. Electron microscope analysis of heteroduplex molecules formed between HPV32 and HPV42 DNAs showed paired regions over about 60 and 87% of their genome lengths under stringent ( T m-18°) and nonstringent ( T m-42°) conditions, respectively. The 6.8-kb HPV DNA and HPV39 DNA formed paired regions over about 63 and 95% of the 6.8-kb fragment length at T m-18° and T m-26°, respectively. These data point to greater DNA sequence homologies than anticipated from the percentages of nuclease S1 resistance. Heteroduplex mapping has allowed the alignment of the physical maps of HPV39 and 42 DNAs and of the 6.8-kb HPV DNA with the map of the open reading frames of the HPV16 genome. So far, HPV42 has been detected only in benign genital lesions showing usually no cell atypia. HPV39 has been detected in a few cases of intraepithelial neoplasias and invasive carcinomas of the uterine cervix. The viral DNA sequences have been found integrated into the cell genome in all four HPV39-associated cervical cancers of our series. It seems most likely that HPV42 belongs to the low-risk group of genital HPVs, while HPV39 represents a potentially oncogenic genital HPV type.

A vaccinia virus DNase preparation which cross-links superhelical DNA.

Multiple DNA-dependent enzyme activities have been detected in highly purified preparations of a single-strand-specific nuclease from vaccinia virus. These enzyme preparations were extensively purified and characterized by using superhelical DNAs as substrates. In particular, the nuclease activity was monitored by the extent of conversion of supercoiled closed duplex DNA (DNA I) to nicked circular DNA (DNA II), which could subsequently be converted to duplex linear DNA (DNA III) by prolonged incubation with the enzyme. DNA species which were not substrates for the enzyme included relaxed closed duplex DNA, DNA II which had been prepared by nuclease S1 treatment or by photochemical nicking of DNA I, and DNA III. With plasmid pSM1 DNA as substrate, the extent of cleavage of DNA I to DNA II was found to increase with superhelix density above a threshold value of about -0.06. The linear reaction products were examined by gel electrophoresis after restriction enzyme digestion of the DNAs from plasmids pSM1 and pBR322 and of the viral DNAs from bacteriophage phi X174 (replicative form) and simian virus 40, and the map coordinate locations of the scissions were determined. These products were further examined by electron microscopy and by gel electrophoresis under denaturing conditions. Electron micrographs taken under partially denaturing conditions revealed molecules with terminal loops or hairpins such as would result from the introduction of cross-links at the cutting sites. These species exhibited snapback renaturation. The denaturing gel electrophoresis experiments revealed the appearance of new bands at locations consistent with terminal cross-linking. With pSM1 and pBR322 DNAs, this band was shown to contain DNA that was approximately twice the length of a linear single strand. The terminal regions of the cross-linked linear duplex reaction products were sensitive to nuclease S1 but insensitive to proteinase K, suggesting that the structure is a hairpin loop not maintained by a protein linker. A similar structure is found in mature vaccinia virus DNA.

Involvement of single-strand breaks in complex formation between single-stranded DNA and nucleoids of Bacillus subtilis.

RNase-unfolded chromosomes of competent Bacillus subtilis are able to take up single-stranded homologous donor DNA fragments in vitro to form donor-recipient DNA complexes (Van Randen and Venema 1981). The unfolded chromosomes behave as supercoiled DNA molecules. X-irradiation increased the formation of unstable and stable complexes between donor and recipient DNA during incubation at 37 degrees C. The complex-forming ability of the unfolded chromosomes increased linearly with increasing X-ray dose, even after complete relaxation of the unfolded chromosomes had occurred. Limited DNase I action increased the complex-forming ability of the chromosomes as effectively as X-irradiation. Unstable donor-recipient DNA complexes can be distinguished from stable ones by their dissociation upon density gradient centrifugation in CsCl at pH 11.2. They are stable at pH 10 (Van Randen et al. 1982a). At an intermediate pH value during isopycnic centrifugation, a fraction of the unstable complexes were stable, suggesting that a range of stabilities existed among the unstable complexes. The donor moiety of the stable donor-recipient DNA complexes was far more resistant to nuclease S1 treatment than that of the unstable ones.

7S-K nuclear RNA from simian virus 40-transformed cells has sequence homology to the viral early promoter.

Previous findings in our laboratory have identified a specific small nuclear RNA (7S-K) that promotes transcription initiation by RNA polymerase II in isolated mammalian nuclei. The present study was designed to investigate the homology between 7S-K RNAs and host and viral sequences in simian virus 40 (SV40)-transformed and in untransformed mouse 3T3 cells, with the object of testing the hypothesis that these RNAs take part in the transcription initiation complex by base pairing to promoter/enhancer regions of active genes. DNA . RNA hybridization experiments, using either Southern or RNA blotting techniques, indicated that both 7S-K and 7S-L RNAs hybridize to midrepetitive fractions of the mouse genome. However, only 7S-K RNA from transformed cells hybridized to SV40 DNA. Restriction mapping and nuclease S1 treatment of the hybridized region of SV40 yielded a 45-nucleotide-long hybrid duplex. Partial sequence analysis of the 5' end of the DNA in this duplex revealed sequence homology with the 21-base-pair repeat sequence, identified as the SV40 early promoter. Because the viral early gene is expressed in transformed cells, we conclude that 7S-K RNAs in these cells contain a species that has sequence homology to a promoter of an active gene. Taking these results together with previous ones, we postulate that the observed stimulatory activity of 7S-K RNA on transcription initiation is due to its recognition of promoter sequences, either to facilitate the formation or as part of the transcription initiation complex.

"Nonrandom" DNA sequence analysis in bacteriophage M13 by the dideoxy chain-termination method.

We describe a rapid "nonrandom" DNA sequence analysis procedure that facilitates the nucleotide sequence determination of large contiguous regions of DNA. The method consists of cloning a restriction endonuclease fragment of interest into bacteriophage M13 followed by construction of a series of nuclease BAL-31 deletion mutants originating from a single site in M13 that is close to the DNA insert. Determination of the size of the deletion mutant is accomplished by hybridization to a complementary single-stranded probe derived from M13 containing that total insert followed by nuclease S1 treatment. Single-stranded M13-insert DNAs of progressively smaller sizes are isolated and analyzed by using a site-specific M13 DNA primer and the dideoxy chain-termination method. In this way, analysis of the DNA sequence proceeds from one end of the total insert to the other in a nonrandom fashion due to generation of a controlled overlapping set of deletion mutants.

Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs

Physical, transcriptional, and translational maps of an EcoRI fragment located between 15,800 and 20,600 base pairs from the left end of the vaccinia virus genome were prepared. Major polypeptides with molecular weights of 14,000 (14K polypeptide), 32,000 and 38,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA made in the presence of cycloheximide and selected by hybridization to lambda recombinant DNA containing the EcoRI fragment. With early RNA made in the presence of cytosine arabinoside, an inhibitor of DNA replication, the polypeptide pattern was similar except for quantitative differences in which less 38K polypeptide was detected as a translation product. With late RNA, isolated 6 h after infection without inhibitors, only traces of the early translation products were found and a new 40K polypeptide was detected. The size of the mRNA's for the 14K, 32K, and 38K polypeptides were determined to be approximately 760,880, and 1,150 nucleotides, respectively, by several independent procedures. Several large early RNAs not shown to code for any additional translation products were also detected. The size of the late message for the 40K polypeptide varied from 920 to 3,100 nucleotides. This heterogeneity appeared to be a general property of vaccinia virus late mRNA's. No evidence of RNA splicing was obtained by analysis of RNA-DNA hybrids after nuclease S1 treatment. Further analyses using separated recombinant DNA strands and restriction fragments indicated that all mRNA's were encoded by the leftward-reading DNA strand and at least two were overlapping. Since early and late mRNA's were encoded by the same DNA strand, the possibility of temporal regulation by transcriptional strand switching was eliminated. In conjunction with previous studies, a transcriptional map of the left 20,600 base pairs of the vaccinia virus genome was derived.

Assumed initiation site of fibroin gene transcription.

To map the initiation site of fibroin gene transcription on cloned genomic fibroin DNA (pFb29), we looked for precursors of fibroin mRNA, first by applying the hybridization mapping method to the RNA from the posterior silk gland of Bombyx mori. The 6.2-kilobase (kb) DNA fragment used for the assay, from 5.7 kb upstream to 0.5 kb downstream from the cap site, was end-labeled at the Xho I site (position +514) within the intervening sequence of the fibroin gene. By analyzing the labeled DNA protected by the RNA from nuclease S1 treatment, we have shown that the intervening sequence is transcribed together with the surrounding gene sequences in the posterior silk gland (producer) but not in the middle silk gland (nonproducer). Large transcripts that could include entire intervening (1 kb) and fibroin mRNA (16 kb) sequences were found. The 5' end of such transcripts was located around or near the cap locus. Synthesis and analysis of the DNA complementary to the transcripts primed within the intervening sequence region also confirmed that the 5' end of the transcripts is near the cap locus. These results strengthen the possibility that the cap site is the transcription initiation site of fibroin gene.

Modification of DNA by the benzo[a]pyrene metabolite diol-epoxide r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

The structural modification of double-stranded circular DNA of simian virus 40 and plasmid ColE1 by in vitro binding of r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene was studied. Stepwise hydrolysis with endonuclease S1 and DNase followed by DNA base analysis by thin-layer chromatography provided evidence that binding to adenine caused the local denaturation of DNA, whereas the more than 10-fold greater binding to guanine did not create such local denaturation. Of the two synthetic double-stranded polymers, poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC), bound to the diol-epoxide, only the former showed a marked hydrolysis after endonuclease S1 treatment, whereas binding occurred 24-fold more on the latter.

Diagnosis and Treatment of Patients with High Hematocrit.

s1 May 1969Diagnosis and Treatment of Patients with High Hematocrit.Karl Tornyos, M.D., F.A.C.P.Karl Tornyos, M.D., F.A.C.P.Search for more papers by this authorAuthor, Article, and Disclosure Informationhttps://doi.org/10.7326/0003-4819-70-5-1086_3 SectionsAboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinkedInRedditEmail ExcerptIn patients with hematocrit of 60% or above the A blood viscosity is increased, and the tissue oxygenation is compromised. There is an increased incidence of cardiovascular complications. Immediate phlebotomy is often done, but if the red cell volume is not increased and the plasma volume and total blood volume are decreased, phlebotomy may be harmful. Twenty-five patients with hematocrits of 60% or above have been studied. Patients with clinical or laboratory evidence of dehydration are not included. Red cell volume was determined by chromium-51 and plasma volume by iodine-131 human serum. The expected red cell volume and plasma volume... This content is PDF only. To continue reading please click on the PDF icon. Author, Article, and Disclosure InformationAffiliations: Charleston, South Carolina PreviousarticleNextarticle Advertisement FiguresReferencesRelatedDetails Metrics 1 May 1969Volume 70, Issue 5Page: 1086-1087KeywordsBloodBlood cellsBlood volumeClinical laboratoriesHematocritPatientsPhlebotomyPlasma cellsPlasma volumeResearch laboratories ePublished: 1 December 2008 Issue Published: 1 May 1969 PDF downloadLoading ...