http://www.alliot.fr/BIO/PDF/Southern_1975.pdf

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Construction and characterization of a class of multicopy plasmid cloning vehicles containing the replication system of miniplasmid P15A are described. The constructed plasmids have cleavage sites within antibiotic resistance genes for a variety of commonly employed site-specific endonucleases, permitting convenient use of the insertional inactivation procedure for the selection of clones that contain hybrid DNA molecules. Although the constructed plasmids showed DNA sequence homology with the ColE1 plasmid within the replication region, were amplifiable by chloramphenicol or spectinomycin, required DNA polymerase I for replication, and shared other replication properties with ColE1, they were nevertheless compatible with ColE1. P15A-derived plasmids were not self-transmissible and were mobilized poorly by Hfr strains; however, mobilization was complemented by the presence of a ColE1 plasmid within the same cell.

Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid.

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

http://stacks.cdc.gov/view/cdc/29899/cdc_29899_DS1.pdf

DNA targeting specificity of RNA-guided Cas9 nucleases

https://science.sciencemag.org/content/sci/160/3825/272.full.pdf

Specific Gene Amplification in Oocytes Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA

https://www.cell.com/cell/pdf/0092-8674(80)90385-2.pdf

A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3′ border of the region

RNA synthesis during the development of Xenopus laevis, the South African clawed toad

Few new ribosomes appear in the cytoplasm of embryos of Rana pipiens or Xenopus laevis (the South African \"clawed toad\") before the tail bud stage.1 At this time the amount of cytoplasmic ribosomes begins to increase; this rise is correlated with an increase of protein in the high speed supernatant fraction as well as with the first appearance or increase of many enzymes. Soon after these events, the embryos develop a requirement for magnesium ions in the medium. Magnesium-starved embryos characteristically stop growing in length and die at early swimming stages (Shumway2 stages 21-23 for Rana pipiens,I or Nieuwkoop-Faber3 stage 40 for Xenopus laevis4). The magnesium requirement coincides with the onset of intense ribosome synthesis and presumably is based on the important role of magnesium ions in maintaining the integrity of the functional ribosome particle. The study of ribosome synthesis during amphibian development has been extended utilizing the lethal anucleolate mutant of Xenopus laevis first described by Elsdale et al.5 These workers discovered a heterozygote mutant with only one nucleolus (1-nu) in each cell, whereas wild-type Xenopus laevis have two nucleoli (2-nu) in the majority of their diploid cells. The progeny resulting from the mating of two heterozygotes (1-nu) fall into three groups having two, one, or zero nucleoli per cell. The ratio of these genotypes is 1:2:1, respectively,5' 6 as expected of a typical Mendelian factor. The heterozygotes (1-nu) lack a secondary constriction (\"nucleolar organizer\") on one of two homologous chromosomes in diploid cells;7 the two comparable chromosomes of the anucleolate homozygous mutants (0-nu) both lack this secondary constriction. The anucleolate mutant (0-nu) has numerous small nucleolar \"blobs\" instead of typical nucleoli, and both nuclear and cytoplasmic RNA have been shown histochemically to be lower in 0-nu embryos after hatching than in controls (1-nu and 2-nu).8 Development of 0-nu embryos is first retarded shortly after hatching.5' 9 The mutant embryos become microcephalic and oedematous and die as swimming tadpoles before feeding. It was apparent that magnesium-starved embryos were to some extent phenocopies of the homozygous mutants (0-nu) since retardation of embryogenesis and growth occurred in both groups of embryos at about the same developmental stage (Fig. 1). The above data, as well as recent studies relating nucleolar function to ribosome synthesis, suggested that the anucleolate mutant might be incapable of synthesizing ribosomes and ribosomal RNA. Material and Methods.-Radioactivity was introduced into developing embryos by incubation with C1'02 at pH 6.0.10 The methods for measuring ribosome and DNA contents have been described previously.' Total RNA was isolated from frozen embryos after homogenization in 0.1 M sodium acetate pH 5.0 containing 4 ,ug/ml polyvinyl sulfate (a ribonuclease inhibitor prepared synthetically by the method of Bernfeld et al.)\" and 0.5% sodium lauryl sulfate (Mann Research Co.). The homogenate was shaken for 5-10 min at 0C with an equal vol of phenol. Nucleic acids were precipitated from the aqueous phase with 2 vol of ethanol and 0.1 vol of M

https://www.pnas.org/content/pnas/51/1/139.full.pdf

Donald D. Brown

Papers

Specific Gene Amplification in Oocytes Oocyte nuclei contain extrachromosomal replicas of the genes for ribosomal RNA

A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3′ border of the region

RNA synthesis during the development of Xenopus laevis, the South African clawed toad

Absence of ribosomal rna synthesis in the anucleolate mutant of xenopus laevis.

Nucleotide sequences in Xenopus 5S DNA required for transcription termination