Dideoxy fingerprinting (ddE): a rapid and efficient screen for the presence of mutations.

We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP.     

Expression of an enhancer-binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus IE1 gene.

The baculovirus Autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. To begin to identify proteins that interact with hr5, DNA-protein interactions were analyzed by using extracts from Spodoptera frugiperda cells and a fragment of DNA containing the left half of the hr5 enhancer. This 252-bp DNA fragment contains two copies of a 30-bp direct repeat (DR30) and two copies of a 24-bp imperfect palindrome contained within a 60-bp direct repeat (DR60). Extracts prepared from normal S. frugiperda cells and cells transfected with pUC8 lacked enhancer-binding proteins. However, when gel shift assays were performed with extracts from cells transfected with a plasmid containing the viral trans-activator IE1 gene, two DNA-protein complexes were formed. Both DNA-protein complexes were specifically inhibited by competition with a 60-bp oligonucleotide corresponding to DR60 but not by competition with a different oligonucleotide corresponding to DR30. Formation of the two complexes did not appear to involve cooperative interactions between binding proteins. When DR60 was used as a probe, a single complex was formed. To measure the enhancer activity of DR60, a reporter plasmid was constructed that contained DR60 cloned upstream of the reporter chloramphenicol acetyltransferase gene under the control of the delayed-early 39K promoter. Transient expression analysis indicated that the oligonucleotide increased expression of this gene 300-fold over the level obtained in the absence of any enhancer sequences.     

Monoclonal Antibodies against Apple 1-Aminocyclopropane-l-Carboxylate Synthase

1-Aminocyclopropane-l-carboxylate (ACC) synthase from applefruits was purified over 5,000-fold by conventional column chromatography.By immunizing mice with this partially purified enzyme preparation,8 hybridoma lines producing monoclonal antibodies against appleACC synthase were isolated. While all 8 clones immunoprecipitatednative ACC synthase, only two clones recognized the putative(48 kDa) ACC synthase on Western blots. When a partially purifiedACC synthase preparation was incubated with S-adenosyl-L-[carboxyl-¹⁴C]methionine(AdoMet), only one radioactive protein of 48 kDa was detectedon sodium dodecyl sulfate-poly-acrylamide gel electrophoresis.This radioactive protein was specifically immunoprecipitatedby the monoclonal antibodies, indicating that apple ACC synthaseis specifically radiolabeled by its substrate AdoMet, as istomato ACC synthase. Thus, the monoclonal antibodies recognizedboth native and AdoMet-inactivated forms of ACC synthase. Whilethese antibodies failed to im-munoprecipitate ACC synthase isolatedfrom ripe tomato fruits, ripe avocado fruits or auxin-treatedmungbean hypocotyls, they were effective in immunoprecipitatingthe enzyme isolated from ripe pear fruits. (Received August 11, 1990; Accepted October 17, 1990)     

Simultaneous identification of estrogen and progesterone receptors by HPLC using a double isotope assay.

Polymorphism of estrogen (ER) and progestin receptors (PR) was analyzed simultaneously using high performance hydrophobic interaction chromatography (HPHIC). HPHIC was used previously to characterize four ER isoforms [Hyder et al., J. Chromat. 397 (1987) 251] based on retention times on Synchropak propyl (100 x 6 mm) HPLC columns (Synchrom, Inc.). ER and PR were prepared from human breast cancer. ER was labeled with 3 nM of either [3H]estradiol-17 beta ([3H]E) or [125I]iodoestradiol-17 beta ([125I]E) while PR was associated with 5 nM of either [3H]R5020 ([3H]R) or [125I]iodovinylnortestosterone ([125I]V). ER was resolved by HPHIC into isoforms MI (Rt = 11 min), I(Rt = 16 min), and II (Rt = 24 min). Isoforms I and II each accounted for ca 45% of specific binding. PR separated into isoforms MI (Rt = 14 min) and I (Rt = 21 min, 80% of specific binding) when eluted with the same gradient used for ER chromatography. Upon inclusion of 10 mM molybdate ER resolved into isoforms MI and MII (Rt = 16 min) and PR into isoforms MI and I (here however isoform MI represented 80-95% of specific binding). Elution patterns were preserved with different batches of stationary phase suggesting the integrity of the isoform distribution. HPLC profiles of ER isoforms labeled with earlier [125I]E or [3H]E were identical as were PR isoform profiles labeled with either [3H]R or [125I]V. Pairs of 125I- and 3H-labeled ligands were used in either combination to monitor ER and PR profiles simultaneously. Isoforms analyzed in 50 biopsies gave reproducible retention times, however the ratio between I and II for ER and MI and I for PR varied. This method allows rapid, simultaneous monitoring of the chromatographic behavior of ER and PR isoforms or other associating proteins or nucleotides. One may now better elucidate their interrelationship as it relates to the hormone-response mechanism.     

石灰菌代谢产物的化学研究(Ⅰ)

从石灰菌(Lactarius hysginus Fr.)提取物的中性部分,分离得到六个化合物,经物理常数和光谱分析,鉴定为麦角甾醇(A)、硬脂酸甲脂(B)、N-苯基-2-萘胺(C)、24E-麦角甾7,22-二烯-6-酮一3β,5α-二醇(D)、庚酰胺(E)、24E-麦角甾-7.22-二烯-3β,5α,6β-三醇(F),其中化合物D作为天然产物尚未见文献报道。     

黄淮海平原高产田作物群体结构特征

本文利用实测资料分析了高产田作物群体结构特征。精播高产栽培麦田春季最大蘖数12×10~6/ha,群体最大叶面积指数5.5—6.0,成穗4.5—5.25×10~6/ha。传统高产栽培麦田春季最大蘖数15—18×10~6/ha,群体最大叶面积指数6.0—6.5,成穗6.0—7.5×10~6/ha。小麦营养生长与生殖生长期叶日积比,精播高产栽培麦田是1∶0.89,传统高产栽培麦田为1∶0.73。夏玉米种植密度主要受叶倾角的影响。紧凑型玉米品种的叶倾角大于65°,种植密度7.5—8.25×10~4/ha;平展型玉米品种的叶倾角小于50°,种植密度5.25—6.0×10~4/ha。     

Macrophage oxidation of L-arginine to nitrite and nitrate: nitric oxide is an intermediate

Previous studies have shown that murine macrophages immunostimulated with interferon gamma and Escherichia coli lipopolysaccharide synthesize NO2-, NO3-, and citrulline from L-arginine by oxidation of one of the two chemically equivalent guanido nitrogens. The enzymatic activity for this very unusual reaction was found in the 100,000g supernatant isolated from activated RAW 264.7 cells and was totally absent in unstimulated cells. This activity requires NADPH and L-arginine and is enhanced by Mg2+. When the subcellular fraction containing the enzyme activity was incubated with L-arginine, NADPH, and Mg2+, the formation of nitric oxide was observed. Nitric oxide formation was dependent on the presence of L-arginine and NADPH and was inhibited by the NO2-/NO3- synthesis inhibitor NG-monomethyl-L-arginine. Furthermore, when incubated with L-[guanido-15N2]arginine, the nitric oxide was 15N-labeled. The results show that nitric oxide is an intermediate in the L-arginine to NO2-, NO3-, and citrulline pathway. L-Arginine is required for the activation of macrophages to the bactericidal/tumoricidal state and suggests that nitric oxide is serving as an intracellular signal for this activation process in a manner similar to that very recently observed in endothelial cells, where nitric oxide leads to vascular smooth muscle relaxation [Palmer, R. M. J., Ashton, D. S., & Moncada, S. (1988) Nature (London) 333, 664-666].     

Monoclonal antibodies specific for type 3 protein kinase C recognize distinct domains of protein kinase C and inhibit in vitro functional activity

Monoclonal antibodies (mAbs) which distinguish Type 3 protein kinase C (PKC) from Types 1 and 2 have been obtained from mice immunized with purified Type 3 PKC from rabbit brain cytosol. Most of these mAbs (seven out of eight) selectively recognize Type 3 versus Types 1 and 2 PKC in both enzyme-linked immunosorbent and immunoblot assays. Trypsin treatment of Type 3 PKC reduced the immunoreactivity with 82-kDa PKC and generated immunoreactive fragments of 45 and 35 kDa. The mAbs can be divided into two classes based on their ability to recognize the 45-kDa catalytic fragment (5/8) or the 35 kDa regulatory domain fragment (3/8). Each of the mAbs inhibits phosphorylation of histone or lipocortin by PKC, although the extent of the inhibition varied. Only those mAbs that recognize the 35-kDa regulatory domain inhibited phorbol ester binding. The inhibition of both kinase and binding activities by this group of mAbs was sensitive to the concentration of phospholipid used in the assay. This functional inhibition suggests that these mAbs may be useful for defining the phospholipid binding domain(s) of Type 3 PKC. The mAbs recognized 82-kDa PKC in a variety of cell types; the presence of smaller molecular weight fragments was not consistently found. Distinct immunofluorescence staining patterns were observed with mAbs directed toward different epitopes, suggesting that there may be heterogeneity in the subcellular localization of PKC. The type specificity of these mAbs will make them valuable tools for studying activation and regulation of Type 3 PKC in cell culture model systems.