Site-specific cleavage of RNA and DNA by complementary DNA--bleomycin A5 conjugates

Bleomycin displays clinical chemotherapeutic activity, but is so nonspecifically toxic that it is rarely administered. It was therefore of interest to determine whether bleomycin could be directed to cleave RNA or DNA at a specific site by conjugation to a complementary oligonucleotide. A 15 nt MYC complementary oligodeoxynucleotide (HMYC55) bearing a 5' bleomycin A5 (Blm) residue was designed to base-pair with nt 7047-7061 of human MYC mRNA. Reactivity of the Blm-HMYC55 conjugate (and mismatch controls) with a MYC mRNA 30-mer, a MYC DNA 30-mer, and a MYC 2'-O-methyl RNA 30-mer, nt 7041-7070, was analyzed in 100 microM FeNH(4)SO(4), 50 mM beta-mercaptoethanol, 200 mM LiCl, 10 mM Tris-HCl, pH 7.5, at 37 degrees C. Cleavage of the substrate RNA or DNA occurred primarily at the junction of the complementary DNA-target RNA duplex, 18-22 nt from the 5' end of the RNA. Reaction products with lower mobility than the target RNA or DNA also formed. Little or no reaction was observed with more than three mismatches in a Blm-oligodeoxynucleotide conjugate. Neither the short RNA or DNA cleavage fragments nor the low mobility products were observed in the absence of Fe(II), or the presence of excess EDTA. The target RNA was also cleaved efficiently by bleomycin within a hybrid duplex with a preformed single-nucleotide bulge in the RNA strand. New Blm-oligodeoxynucleotide conjugates containing long hexaethylene glycol phosphate based linkers between oligodeoxynucleotide and bleomycin were designed to target this bulge region. These conjugates achieved 8-18% cleavage of the target RNA, depending on the length of the linker. Blm-oligodeoxynucleotide conjugates thus demonstrated sequence specificity and site specificity against RNA and DNA targets.     

Heat shock cognate protein 70 is involved in rotavirus cell entry

In this work, we have identified the heat shock cognate protein (hsc70) as a receptor candidate for rotaviruses. hsc70 was shown to be present on the surface of MA104 cells, and antibodies to this protein blocked rotavirus infectivity, while not affecting the infectivity of reovirus and poliovirus. Preincubation of the hsc70 protein with the viruses also inhibited their infectivity. Triple-layered particles (mature virions), but not double-layered particles, bound hsc70 in a solid-phase assay, and this interaction was blocked by monoclonal antibodies to the virus surface proteins VP4 and VP7. Rotaviruses were shown to interact with hsc70 at a postattachment step, since antibodies to hsc70 and the protein itself did not inhibit the virus attachment to cells. We propose that the functional rotavirus receptor is a complex of several cell surface molecules that include, among others, hsc70.     

Key-factor analysis of immature stages of Aedes scapularis (Diptera: Culicidae) populations in southeastern Brazil

Development times and mortality factors were investigated for populations of Aedes scapularis (Rondani) occurring in natural temporary ground pools in southeastern Brazil. Analysis of life tables by the key-factor method showed that mortality caused by desiccation of breeding sites was the principal factor accounting for fluctuations in population sizes throughout the study period. For cohorts that completed development, mortality attributed to predation by aquatic insects ranged from 68 to 96% and was the most important cause of death. In these cohorts, final population size was determined by the high mortality rates that occurred during the fourth larval instar and pupal stages; however, the key-factor best accounting for the population fluctuations was predation of fourth instar larvae. The short mean time of 7.1 days for synchronous development of the larvae and the lack of evidence of density-dependent mortality suggested that the A. scapularis populations were not strongly regulated and can attain relatively high densities.     

Numerical Taxonomy of Microorganisms Isolated from Goat Cheese Made in Chile

118 strains of heterotrophic microorganisms were isolated from goat cheese produced domestically in the IV Region of Northern Chile (Serene, Ovalle, and Illapel) and sold in supermarkets in Valparaíso, Chile. The results of 89 phenotypic tests were numerically analyzed against 17 reference strains, using the simple matching coefficient (S_SM). Thirteen phena were found at a 78% similarity level. Five of them (A, B, C, D, and E) were assigned to the family Enterobacteriaceae, phenon F was identified as belonging to the genus Aeromonas and strains of phenon G were assigned to the genus Acinetobacter. The other phena were identified as being members of the genera Bacillus (H, I, and J), Staphylococcus (K), Enterococcus (L), and Micrococcus (M). Approximately 19% of the isolates were Escherichia coli and 27%, Staphylococcus aureus. Received: 20 February 2001 / Accepted: 12 April 2001     

Enhancement of chloroplast energization in Chlorella by treatments inactivating the nitrate-reducing system

Inactivation of the nitrate-reducing system in whole cells of Chlorella vulgaris Bejerinck by darkening, nitrogen starvation, ammonium, or cycloheximide brings cells into a state with a high yield of the millisecond-delayed fluorescence of chlorophyll. Activation of this system by illumination, by adding glucose to dark-adapted cells or nitrate to nitrogen-starved cells brings the cells into a low-yield state. The transitions between the lowand high-yield state induced by alternating light and dark periods are suppressed by tungstate and restored by subsequent molybdate addition. The drop in the delayed-fluorescence yield upon activation of the nitrate-reducing system is associated with the decrease of the amplitude of the electrochemical proton gradient across the thylakoid membrane of the chloroplast, as evidenced by the kinetics of the light-induced adsorption changes at 520 nm. The decrease of the proton gradient may be caused by the electron flow diverting from the cyclic path in photosystem I as a result of the activation of the electron transfer from ferredoxin to nitrite.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Floristic groups and plant communities of southeastern Santa Fe,Argentina

This paper is a survey of the vegetation of the southeastern departments in the Province of Santa Fe (Argentina). The vegetation was analyzed following Braun-Blanquet's approach modified by Mueller-Dombois & Ellenberg (1974). The most relevant species of the region were placed in 25 groups according to their requirements or general behaviour. Most of the communities are herbaceous, and apart from the woody and some other miscellaneous ones they were grouped into three ecologically and floristically defined sets. The first set, the Stipa grasslands and related communities, which are characterized by the more or less abundant presence of Stipa hyalina, Stipa neesiana and Stipa papposa, comprises five different communities. The second, the halophilous communities, comprises five communities, the two Spartina ssp. grasslands, the halophilous prairies of Distichlis spicata, the short sedge Scirpus americanus communities and the ‘pela-dales’. The third, the hygrophilous communities, comprises nine communities which are not so well defined as the ones in the other sets. Besides, two further communities have been included, the Paspalum quadrifarium and the Melica macra tall grasslands.     

Whole-genome sequencing and assembly with high-throughput, short-read technologies

While recently developed short-read sequencing technologies may dramatically reduce the sequencing cost and eventually achieve the $1000 goal for re-sequencing, their limitations prevent the de novo sequencing of eukaryotic genomes with the standard shotgun sequencing protocol. We present SHRAP (SHort Read Assembly Protocol), a sequencing protocol and assembly methodology that utilizes high-throughput short-read technologies. We describe a variation on hierarchical sequencing with two crucial differences: (1) we select a clone library from the genome randomly rather than as a tiling path and (2) we sample clones from the genome at high coverage and reads from the clones at low coverage. We assume that 200 bp read lengths with a 1% error rate and inexpensive random fragment cloning on whole mammalian genomes is feasible. Our assembly methodology is based on first ordering the clones and subsequently performing read assembly in three stages: (1) local assemblies of regions significantly smaller than a clone size, (2) clone-sized assemblies of the results of stage 1, and (3) chromosome-sized assemblies. By aggressively localizing the assembly problem during the first stage, our method succeeds in assembling short, unpaired reads sampled from repetitive genomes. We tested our assembler using simulated reads from D. melanogaster and human chromosomes 1, 11, and 21, and produced assemblies with large sets of contiguous sequence and a misassembly rate comparable to other draft assemblies. Tested on D. melanogaster and the entire human genome, our clone-ordering method produces accurate maps, thereby localizing fragment assembly and enabling the parallelization of the subsequent steps of our pipeline. Thus, we have demonstrated that truly inexpensive de novo sequencing of mammalian genomes will soon be possible with high-throughput, short-read technologies using our methodology.     

以黑丝羽乌骨鸡为供体制作家鸡嵌合体的研究

以黑丝羽乌骨鸡（BS）为供体,自来航鸡（WL）为受体,进行了BCs嵌合体制作技术研究。结果表明：（1）“黑羽”对“白羽”、“丝羽”对“片羽”为完全隐性,可以在供体与嵌合体测交中,后代是否出现这些特征作为种系嵌合体判断依据。（2）供体的其它表型,对受体属于完全或不完全显性,可以作为体细胞嵌合体判断依据。（3）通过改进嵌合体制作技术,嵌合体雏鸡的出壳率为40％（29／73）,其中据羽色判断的嵌合体率为18％（13／73）;以黑羽为依据选择体细胞嵌合体雏鸡,10只饲养至720d,其中60％（6／10）的嵌合体外观基本不变（其余的换羽后褪去黑羽）;嵌合体鸡与供体测交,以黑羽、灰羽和丝羽判断,8只嵌合体鸡的种系传递率分别为2．5％-71．4％、5．5％～14．3％以及1．7％～10．5％。首次利用BS鸡资源,建立了多表现型嵌合体模型,为家鸡嵌合体技术深入研究提供了方便的检测方法。