Species-Specific Random Amplified Monomorphic DNA

Intra- and interspecific variability of total DNA isolated from haploid megagametophytes of coniferous species was examined using polymerase chain reaction with random primers. Based on this technique, one can with certainty detect heterozygosity at gene loci carrying null alleles and thus reveal cryptic intraspecific genetic variation. Large population samples were used. Along with random amplified polymorphic DNA, i.e., widely known fragments (amplicons) polymorphic within a species, we found invariant loci lacking individual or geographic variability but differentiating species within genera and other taxa. This DNA was termed RAMD (random amplified monomorphic DNA) to distinguish it from polymorphic DNA. Our findings suggest that genetic monomorphism of species and the dual structure of the eukaryotic genome can be detected at the DNA level as was previously shown for protein gene markers.     

Amplified DNA sequences in cancers

Amplification of genes other than known oncogenes was analyzed using an in-gel DNA renaturation method, in which a mixture of restriction fragments of radioactively labelled tracer DNA and unlabelled driver DNA was electrophoresed and amplified DNA fragments were visualized after two cycles of denaturation and renaturation in the gel. Different DNA fragments were found to be amplified more than 400 fold in NB1, a neuroblastoma cell line, in Y79, a retinoblastoma cell line and in H69, a small cell lung carcinoma cell line, in addition to 120 to 160-fold amplification of N-myc gene in these three cell lines.     

Amplified microRNA detection by templated chemistry

MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate. The sequence is amplified through rolling circle amplification with 29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ～50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ～15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.     

Amplified DNA in Streptomyces fradiae.

A spontaneous mutant of Streptomyces fradiae contained an amplifiable unit of DNA with a sequence length of approximately 10.5 kilobases that was amplified to approximately 500 copies per chromosome. The amplified DNA appears to be cryptic. SalI fragments of the amplified DNA were cloned into Escherichia coli to construct a restriction map and characterize the amplified DNA. The amplified DNA contained tandem repeats of the amplifiable unit of DNA. The unit had an average base composition of 71% guanine plus cytosine, similar to the chromosomal DNA of Streptomyces species. At least a portion of the amplifiable unit of DNA was present at a low copy number in the wild-type strain. The phenotype of amplified DNA was designated Ads1SF for amplified DNA sequence 1 in S. fradiae.     

Stains for Strands in Sieve Tubes

Two very simple procedures give a staining-fixation of the so-called “strands” as well as portions of the sieve plates of sieve tubes of various broad-leaved deciduous trees. One procedure consists of placing hand-made sections (radial or tangential) of inner bark for 5 min in a 0.2% solution of ponceau S in 3% trichloroacetic acid, then soaking 5 min in 5% acetic acid. A second procedure consists of placing sections in 0.001% nigrosin in 2% acetic acid for approximately 15 hr, then washing briefly in distilled water. In the former procedure, strands, sieve plates, and what appears to be plasmalemma, appear reddish or pink, while cell walls do not stain. In the latter, strands and sieve plates appear bluish but phloem cell walls also become bluish, although xylem cell walls usually remain unstained.     

The Translocation of Sugar in Sieve Tubes

A theory is put forward to account for the translocation ofsugars in terms of electrokinetic forces developed in the cytoplasmof the sieve plates. A major role is suggested for potassiumas responsible both for the movement of solution and for maintainingthe necessary potentials across the sieve plates. The mechanismfor the latter invokes the active uptake of potassium (or someother ion) by the companion cells. It is suggested that in theoverall process of conduction there are features which can bedescribed as involving the elements of inertia and positiveenergy feedback, and that a possible result of these would belongitudinal oscillations in the phloem columns which mightassume importance as the seat of rhythms and as a backgroundfor photoperiodic effects, and which might be adjusted to animposed period by callose constrictions. The mechanism put forward would seem to be physically adequate even for very lengthyphloem columns, and it would seem possible for it to accommodatemost, if not all, of the major experimental facts. It impliesthe active transport of water, as well as of sugars and otherchemical species.     

A Working Model of a Sieve Tube

A working model of a sieve tube is described, based upon thePressure-Flow Hypothesis. The flow of solution in the sievetube is envisaged as being due to the joint influence of anaxial turgor pressure gradient and a lateral water potentialdifference which causes an intake of water along the tube. Inthis second respect the model differs from that originally suggestedby Münch. The model comprises a length of dialysis tubing rendered semi-permeable,incorporating capillary resistances at regular intervals. Asucrose solution is pumped into one end and collected at theother, the whole being submerged in water. Turgor pressures and concentrations along the model were recordedat intervals; thus the approach to the steady state was followed.In the steady state ¹⁴C-sucrose was introduced to the solutionbeing pumped and its approach to a steady distribution in themodel was followed. Important conclusions reached are that the Munch Pressure-FlowHypothesis implies a pressure profile convex upwards, a velocityincrease down the phloem, and an exponential fall in tracerconcentration without its lateral leakage.     

Filamentous Proteins from Plant Sieve Tubes

PROTEIN filaments are characteristic structural components of the assimilatory conducting elements of angiosperm plants (“P protein” of Cronshaw and Esau¹). We have isolated filamentous structures from the phloem exudate of cut cucurbit stems². The presence of the filaments could be clearly demonstrated after negative staining with the electron microscope.     

Sieve elements caught in the act

Phloem is a puzzling plant tissue owing to the unique natural defence responses of the sieve elements to any kind of mechanical manipulation. Recent non-invasive studies have enabled real-time observation of events in intact sieve tubes, including mass transport, sieve-pore sealing and conformational changes of structural proteins. These studies further highlighted the importance of the symplasmic setting for development and functioning of the sieve elements. Exchange of macromolecules between companion cells and sieve elements is indispensable for the survival of the sieve element, but also seems to be involved in long-distance communication. How the branched plasmodesmata between sieve element and companion cell function as corridors for the passage of macromolecules is an intriguing but unresolved story.     

Amplified stretch of bottlebrush-coated DNA in nanofluidic channels

The effect of a cationic-neutral diblock polypeptide on the conformation of single DNA molecules confined in rectangular nanochannels is investigated with fluorescence microscopy. An enhanced stretch along the channel is observed with increased binding of the cationic block of the polypeptide to DNA. A maximum stretch of 85% of the contour length can be achieved inside a channel with a cross-sectional diameter of 200 nm and at a 2-fold excess of polypeptide with respect to DNA charge. With site-specific fluorescence labelling, it is demonstrated that this maximum stretch is sufficient to map large-scale genomic organization. Monte Carlo computer simulation shows that the amplification of the stretch inside the nanochannels is owing to an increase in bending rigidity and thickness of bottlebrush-coated DNA. The persistence lengths and widths deduced from the nanochannel data agree with what has been estimated from the analysis of atomic force microscopy images of dried complexes on silica.     

新生隐球菌的AFLP分析

目的评估AFLP-DNA指纹技术在新生隐球菌分类中应用情况。方法新生隐球菌基因组DNA用双酶酶切,双链接头连于其酶切末端,用与接头和酶切位点互补的引物扩增DNA片段,其产物在高分辨的变性聚丙酰胺凝胶上电泳分离,然后进行银染。结果分析来自5种血清型和临床分离株的18株新生隐球菌,可见有30多条大小在30～500bp的DNA-AFLP指纹,相同的血清型有不同的指纹图谱,来自同一患者不同病期的两株分离株和来自同一患者患者的不同部位的两株分离株都显示出相同的带型。结论显示了AFLP的高分辨率,是适用于新生隐球菌流行病学调查的有力工具。     

Amplified genes as therapeutic targets in cancer

The most effective targeted cancer therapies have arisen from research into genetically altered oncogenes, including BCR-ABL, HER2, RAS and EGFR. Recent advances in cancer genetics have identified many regions of the genome that undergo amplification (increase in copy number) but, in most cases, the key oncogenic targets driving the growth and survival of cancer cells remain unknown. In this review, we discuss high-throughput technologies for the discovery of putative oncogenes, and clinical and functional validation of these genes as targets for therapy. New technologies in translational genomics facilitate the identification, validation and prioritization of candidate molecular targets for anti-cancer therapy.     

大鼠RAPD标记的观察

采用随机扩增多态ＤＮＡ（ＲＡＰＤ）技术,分析ＳＤ和Ｗｉｓｔａｒ二种大鼠的基因多态性,探讨用ＲＡＰＤ标记鉴别二种大鼠及其血标本实验中的认证,结果表明,二种大鼠表现出了各自不同的多态性ＲＡＰＤ标记,作为大鼠的分子标记,可在基因水平区别二种大鼠,故认为是一种大鼠研究的分子依据。