Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.     

猪胰脏细胞cDNA文库的构建

 本文以新鲜猪胰脏为材料,采用异硫氰酸胍法和寡聚(dT)-纤维素亲和层析法,提取了Poly(A)~+RNA,经麦胚无细胞体系鉴定其体外翻译活性,~3H-Leu参入量为空白对照的5倍。参照Gubler和Hoffman等人的方法,以此poly(A~+)RNA为模板,合成总cDNA,并采用多聚物加尾法,与pUC19质粒重组,转化入感受态E.coliJM107,进行分子克隆,其转化率为3.6×10~4克隆子/μgcDNA。并对重组质粒DNA进行了酶切鉴定。     

Characterization of a soybean cDNA clone encoding the mitochondrial isozyme of aspartate aminotransferase,AAT4

A soybean leaf cDNA clone, pSAT2, was isolated by hybridization to a carrot aspartate aminotransferase (EC 2.6.1.1.; AAT) cDNA clone at low stringency. pSAT2 contained an open reading frame encoding a 47640 Da protein. The protein encoded by pSAT2 showed significant sequence similarity to AAT proteins from both plants and animals. It was most similar to two Panicum mitochondrial AATs, 81.5% and 82.0% identity. Alignment of the pSAT2-encoded protein with other mature AAT enzymes revealed a 25 amino acid N-terminal extension with characteristics of a mitochondrial transit peptide. A plasmid, pEXAT2, was constructed to encode the mature pSAT2 protein lacking the putative mitochondrial transit peptide. Escherichia coli containing the plasmid expressed a functional AAT isozyme which comigrated with the soybean AAT4 isozyme during agarose gel electrophoresis. Equilibrium sucrose gradient sedimentation of soybean extracts demonstrated that AAT4 specifically cofractionated with mitochondria. Antibodies raised against the pEXAT2-encoded AAT protein reacted with AAT4 of soybean and not with other AAT isozymes detected in soybean tissues, providing further evidence that clone pSAT2 encodes the soybean mitochondrial isozyme AAT4.     

Ribosomal proteins are encoded by single copy genes in Dictyostelium discoideum

Five recombinant plasmids which encode ribosomal proteins (r-proteins) from Dictyostelium discoideum have been isolated. Poly(A) + RNA was size-fractionated by preparative agarose gel electrophoresis and a fraction encoding proteins of less than 35 kDa was used to construct a cDNA library in the plasmid vector pBR322. Individual clones from the library were screened by hybrid-selected translation and those encoding r-proteins were identified by co-migration of the translation products in two-dimensional gel electrophoresis with marker proteins purified from Dictyostelium ribosomes. Initial characterization using the five cDNA plasmids indicates that these r-proteins are encoded by single copy genes and that they are not tightly clustered in the genome.     

Accelerated screening of cDNA libraries using the polymerase chain reaction and Southern blotting

cDNA libraries are normally constructed in either phage or plasmid vectors and screened for sequences of interest using antibodies or, more commonly, nucleic acid probes. To clone a sequence of interest from a library generally involves at least three rounds of hybridization with ³²P-labeled probes. This approach is highly labor intensive, and no information about the size of the hybridizing insert is obtained until the clones have been purified and the insert DNA analyzed by restriction enzyme digestion. We report on a rapid screening protocol for libraries constructed in bacteriophage lambda vectors involving polymerase chain reaction amplification of the insert from hybridizing phage plaques and on its analysis by agarose gel electrophoresis and Southern blotting. This can take place after only one round of conventional screening, and phage from a large number of positively hybridizing plaques can be analyzed by a “one-tube” reaction.     

黑木耳核糖体失活蛋白cDNA 3′-端的克隆与分析

为了克隆黑木耳核糖体失活蛋白cDNA 3′-端,根据随机测得的中间一段蛋白质序列设计简并引物,应用RT-PCR和3′-RACE反应方法,将得到的基因片段与质粒连接,并转化至大肠杆菌中,进行蓝白筛选,再通过琼脂糖凝胶电泳法和PCR法验证白斑,从而得到阳性重组质粒,最后克隆出该目的蛋白基因片段。结果表明,克隆出的cDNA 3′-端为330bp的开放阅读框(ORF),编码107个氨基酸和2个终止密码子。经试验证明和文献检索,克隆得到的cDNA 3′-端为一种新基因。     

Bacterial Populations Colonizing and Degrading Rice Straw in Anoxic Paddy Soil

Rice straw is a major substrate for the production of methane, a greenhouse gas, in flooded rice fields. The bacterial community degrading rice straw under anoxic conditions was investigated with molecular methods. Rice straw was incubated in paddy soil anaerobically for 71 days. Denaturing gradient gel electrophoresis (DGGE) of the amplified bacterial 16S rRNA genes showed that the composition of the bacterial community changed during the first 15 days but then was stable until the end of incubation. Fifteen DGGE bands with different signal intensities were excised, cloned, and sequenced. In addition, DNA was extracted from straw incubated for 1 and 29 days and the bacterial 16S rRNA genes were amplified and cloned. From these clone libraries 16 clones with different electrophoretic mobilities on a DGGE gel were sequenced. From a total of 31 clones, 20 belonged to different phylogenetic clusters of the clostridia, i.e., clostridial clusters I (14 clones), III (1 clone), IV (1 clone), and XIVa (4 clones). One clone fell also within the clostridia but could not be affiliated to one of the clostridial clusters. Ten clones grouped closely with the genera Bacillus (3 clones), Nitrosospira (1 clone), Fluoribacter (1 clones), and Acidobacterium (2 clones) and with clone sequences previously obtained from rice field soil (3 clones). The relative abundances of various phylogenetic groups in the rice straw-colonizing community were determined by fluorescence in situ hybridization (FISH). Bacteria were detached from the incubated rice straw with an efficiency of about 80 to 90%, as determined by dot blot hybridization of 16S rRNA in extract and residue. The number of active (i.e., a sufficient number of ribosomes) Bacteria detected with a general eubacterial probe (Eub338) after 8 days of incubation was 61% of the total cell counts. This percentage decreased to 17% after 29 days of incubation. Most (55%) of the active cells on day 8 belonged to the genus Clostridium, mainly to clostridial clusters I (24%), III (6%), and XIVa (24%). An additional 5% belonged to the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides phylum, 4% belonged to the α, β, and γ Proteobacteria, and 1.3% belonged to the Bacillus subbranch of the gram-positive bacteria with a low G+C content. The results show that the bacterial community colonizing and decomposing rice straw developed during the first 15 days of incubation and was dominated by members of different clostridial clusters, especially clusters I, III, and XIVa.     

Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the ‘secretome’). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the ‘signal trap’ strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.     

Transcriptional products of the human placental lactogen gene

Poly(A+)RNA from human term placenta was translated in a mouse-derived cell-free system. A major band corresponding to preplacental lactogen (pre-hPL) and a minor band co-migrating with mature hPL represent approximately 15% of the total radioactively labeled proteins. Analysis of the poly(A+)RNA by agarose gel electrophoresis showed a prominent band at approximately 860 nucleotides. A corresponding band was observed in Northern blots of total RNA, hybridized with 32P-labeled recombinant plasmid containing a portion of hPL cDNA. Similar analyses of nuclear RNA showed at least four additional bands at 990, 1200, 1460, and 1760 nucleotides, respectively, which are likely precursors of hPL mRNA. Poly(A+)RNA was also used to construct a cDNA library. Approximately 5% of the clones were found to hybridize to hPL DNA sequences, indicating that hPL mRNA is indeed very abundant in term placental tissue. One recombinant plasmid containing an insert of approximately 815 base pairs was isolated and characterized by restriction enzyme mapping and electron microscopy. Heteroduplexes constructed between the cDNA and the DNA isolated from an hPL genomic clone revealed four small intervening sequences which can account for the lengths observed for the hnRNA molecules.     

人snail真核表达载体构建与鉴定

目的:构建人snail基因真核表达载体并鉴定。方法:使用RT-PCR法获取人snail基因全长c DNA,经Bam H I、Eco R I双酶切、连接,插入pc DNA3.1(+)真核表达载体,转化TOP10感受态细胞,用含氨苄青霉素的LB培养基筛选阳性克隆,提取质粒双酶切电泳及测序鉴定,瞬时转染siha细胞Western-blot从蛋白水平鉴定重组质粒在真核细胞内的表达。结果:pc DNA3.1-snail重组质粒经酶切电泳符合预期片段,测序鉴定插入片段与NCBI Gen Bank文库中人snail序列一致,重组质粒瞬时转染后snail蛋白表达量明显增高。结论:成功构建pc DNA3.1-snail重组质粒载体,为进一步探讨snail基因生物学功能奠定了基础。     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Improved lysis of group N streptococci for isolation and rapid characterization of plasmid deoxyribonucleic acid.

Procedures for effective cellular lysis and plasmid deoxyribonucleic acid (DNA) isolation from group N streptococci were developed. Cells were grown at 32 degrees C for 4 h in a modified Elliker broth containing 20 mM DL-threonine. After cellular digestion with 2 mg of lysozyme per ml for 7 min at 37 degrees C, 1% sodium dodecyl sulfate exposure resulted in complete and immediate lysis. Lactose (Lac) plasmid species in Streptococcus lactis C2 and S. cremoris B1 (30 and 37 megadaltons, respectively) were demonstrated upon examination of DNA from the cleared lysates by agarose gel electrophoresis. Increasing the lysozyme treatment to 20 min or more resulted in loss of the Lac plasmid, whereas other resident plasmids were unaffected and demonstrable in agarose gels. Diethylpyrocarbonate added before lysis prevented Lac plasmid loss in 20-min lysozyme-treated cells, but was not effective after 40 min of lysozyme treatment. The results suggested that endogenous nuclease activity during the lysozyme treatment period initiated Lac plasmid DNA loss. The development of an efficient lysis procedure for the group N streptococci allowed rapid identification and characterization of plasmid DNA by agarose gel electrophoresis. The plasmid composition of S. lactis C2 and S. cremoris B1, as determined by agarose gel electrophoresis, compared favorably to previous electron microscopic observations.     

Plasmid-mediated NAD independence in Haemophilus parainfluenzae.

The location of the genes coding for NAD independence in four unusual clinical isolates of Haemophilus parainfluenzae was determined by transferring these genes to plasmid-free Haemophilus influenzae Rd by transformation and analysing transformants for the presence of plasmids by agarose gel electrophoresis. All NAD-independent transformants were found to carry a single plasmid species. The plasmids, originally harboured by the four H. parainfluenzae isolates recovered from unrelated sources, were of the same size (5.25 kb). Spontaneous reversion to NAD dependence occurred with a low frequency (0.1 to 0.2% of the progeny of a single clone) in both H. parainfluenzae and H. influenzae Rd. The revertants had lost this small plasmid. Mitomycin C exhibited a plasmid 'curing' effect with a frequency of 'curing' of between 1 and 6% of the surviving clones. It was concluded that the genes conferring NAD independence were located on the small 5.25 kb plasmid.     

一种碱裂解菌液直接电泳快速筛选重组子的方法

目的：从碱裂解法提取质粒DNA的原理．经过实验摸索获得一种快速、经济、结果可靠的菌液直接碱裂解电泳筛选重组子的方法。方法：不需提取质粒DNA．只需将细菌培养液碱裂解后直接进行普通琼脂糖凝胶电泳分析,就可以快速筛选出转化重组子。结果：结果和提取质粒酶切鉴定鉴定结果一致。结论：经实验证明菌液直接碱裂解电泳筛选重组子是一种快速、经济、可靠的方法。     

Enzyme variation among clones of Trypanosoma cruzi

The genetic relationships within and between stocks of Trypanosoma cruzi were studied by the in vitro isolation of clones and sub-clones and by the comparison of their isoenzyme patterns in thin-layer starch-gel electrophoresis. Altogether 13 clones and 36 sub-clones were isolated from stocks CL89 and Y207 of T. cruzi. Employing the enzymes L-alanine aminotransferase (ALAT), L-aspartate aminotransferase (ASAT), glucose phosphate isomerase (GPI), malic enzyme (ME), malate dehydrogenase (MDH), glucose-6-phosphate dehydrogenase (G6PD), 6-phosphogluconate dehydrogenase (6PGD), and phosphoglucomutase (PGM), two zymodemes, B and C, emerged among the clones with distinct banding patterns. These zymodemes were consistently distinguished by ALAT, ASAT, GPI, G6PD, 6PGD, and PGM and the differences in enzyme profiles were simultaneously reflected in all six enzyme systems. That the enzymic characters as genetic determinants are stable was demonstrated after recloning and successive replica-platings, i.e., the distinct enzyme patterns remained consistent and homogeneous, the siblings retained the enzyme profiles of their parental clones, and no segregation of the enzyme patterns was observed. Our data from clone and sub-clone examinations show that the isoenzymes act as labels to characterize T. cruzi stocks. Furthermore, enzyme variation was demonstrated among clones isolated from stock CL89.     

Apparent Involvement of a Plasmid in Phaseotoxin Production by Pseudomonas phaseolicola

Three naturally occurring toxigenic strains (HB-36, G-50, and HB-33), one nontoxigenic strain (HB-20), and one ultraviolet light-induced toxinless mutant (G-50 Tox⁻) of Pseudomonas phaseolicola were examined by dye-buoyant density equilibrium centrifugation for the presence of plasmid deoxyribonucleic acid. All strains contained plasmid deoxyribonucleic acid. Comparison of the plasmid deoxyribonucleic acid of different strains by agarose gel electrophoresis showed that strain G-50 harbored three plasmids, whereas the rest of the strains contained two plasmids each. Irrespective of their toxigenicity, all strains shared the large-sized first plasmid band, but differed with respect to other plasmids. Restriction endonuclease analyses of the plasmids indicated that a 22.50-megadalton plasmid was common to two of the toxigenic strains (HB-36 and G-50). However, strain HB-33, which is also toxigenic, contained a much smaller plasmid (4.23 megadaltons). It is hypothesized that this small plasmid may have arisen by a recombination event from a larger plasmid.     

Methods for Inducing Selfing, Selfing and Its Role in the Life Cycle of Euplotes woodruffi syngen 3 (Ciliophora)

Methods for inducing selfing, and the relation between selfing and the life cycle of Euplotes woodruffi syngen 3 are reported. Three intercrossing stocks were used in this experiment. Selfing was induced with several treatments as follows: cell-free fluid from the cultures of complementary mating types; intact cells of GI or S phase in the cell cycle; heat-killed cells, and lysed cells of GI-, S-, and D-phase cells which were prepared by freeze-thawing. Stock SJ-27 was used as a parental stock from which Fl clones were originated through selfing. The other two stocks, SJ-8 and SJ-19, were used as testers. The period of immaturity varied from clone to clone. The heterotypic conjugation of clones with cells of stock SJ-8 seems to occur earlier in the life cycle than with cells of stock SJ-19. This result shows that this syngen has an adolescent period in the life cycle. The length of selfing immaturity seems to be different from that of crossing immaturity, and selfing appeared slightly later than crossing with testers. But the clones in which selfing 1st occurred are considered to be in adolescence or maturity, not in senility. Once selfing appeared in any clone, the clone continued to produce selfing pairs till just before clonal death. The viability of selfing and of outcrossing were compared and found not significantly different. Inbreeding depression took place in some of the F2 clones by successive selfing. The viability of F2 clones from young parents was significantly higher than that from old parents (220 to 230 fissions) both in selfing and outcrossing. The total life spans which were studied in three F1 clones were 168 to 264 fissions.