Primed in situ labelling detection of 5S rDNA sequences in normal and androgenetic rainbow trout

Sex genotypes in mature androgenetic and control rainbow trout Oncorhynchus mykiss were determined using primed in situ labelling (PRINS) detection of 5S rDNA sequences on the X chromosomes. Three sex genotypes, corresponding to phenotypic sex, were revealed: female XX, male XY and supermale YY.     

Prins and C-PRINS: Promising tools for the physical mapping of the lupin genome

Two molecular cytogenetics methods, PRINS (primed in situ DNA labeling) and C-PRINS (cycling PRINS), were optimized for the physical mapping of several types of DNA sequences on the mitotic chromosomes of the narrow-leafed lupin (Lupinus angustifolius L.). The fragment of the FokI element from Vicia faba was localised by indirect PRINS reaction. Two other sequences, fragments of the coding sequences of L. luteus and of L. angustifolius, were localised by indirect C-PRINS. These techniques are faster and more sensitive than FISH, and they allowed the mapping of short DNA fragments. The data obtained shows that both types of PRINS are valuable tools for chromosome identification in lupin.     

人类中期染色体引物原位延伸标记的研究

染色体上引物原位延伸标记在研究染色体结构和基因定位等方面具有重要意义,分别应用随机引物和ＳＯＸ基因兼并引物人类染色体上进行了原位延伸标记,结果表明,随机引物伸在染色体上呈现明暗相间的带纹样特征。ＳＯＸ基因兼并引物延伸发现了更多的ＳＯＸ基因位座,并进一步证实该家族基因在基因组中是散存在的。     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.     

Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

 The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS), a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS, C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection of low-copy DNA sequences. Received: 17 September 1996 / Accepted: 18 October 1996     

Mapping of rRNA genes and telomeric sequences in Danube salmon (<Emphasis Type="Italic">Hucho hucho</Emphasis>) chromosomes using primed in situ labeling technique (PRINS)

In the current paper we described the application of primed in situ (PRINS) labeling approach for the chromosomal mapping of repetitive DNA sequences in Danube salmon (Hucho hucho) (2n = 82, NF = 112). PRINS was successfully performed with primers enabling amplification of 5S rRNA genes (minor rDNAs), NOR building DNA sequences (major rDNAs), and telomeric sequences. Two loci of 5S rRNA were observed on distinct chromosome pairs; the minor arrays were located interstitially on the long (q) arms of two large metacentrics (chromosomes No. 3) and the large clusters of 5S rDNAs were assigned to the short (p) arms of two subtelocentric chromosomes No. 18. Major rDNA clusters were observed on the p-arms of two submeta-subtelocentric chromosomes No. 10. These chromosomal areas were built with GC-rich chromatin what was proved in the course of chromomycin A(3) (CMA(3)) staining performed sequentially. Major and minor rDNA families were not co-localized in the Danube salmon chromosomes.The distinct hybridization signals at the ends of all the chromosomes were provided in the course of PRINS with (CCCTAA)( n ) primer. The chromosomal localization of rRNA genes and telomeric DNA sequences was discussed in the context of Salmonidae karyotype evolution.     

Detection of the protozoan Neospora caninum Using in situ Polymerase Chain Reaction

A new method to detect the protozoan Neospora caninum using indirect in situ polymerase chain reaction (PCR) is described. In situ PCR combines the advantages of the extraordinarily high sensitivity and specificity of PCR and the in situ representation of immunohistochemical methods. We describe an indirect in situ PCR, whereby the amplified products were detected using a primed in situ (PRINS) reaction with hapten-labeled nucleotides and visualized using fluorochrome-labeled antibodies. This technique was carried out in both infected cell cultures and formalin fixed, paraffin embedded tissues. Clear signals were obtained in the N. caninum positive samples using in situ PCR, whereas control slides with Toxoplasma gondii infected tissues always yielded negative results.     

运用PRINS和体细胞杂种克隆板对猪新微卫星的定位

本研究采用引物原位DNA合成PRINS)技术,结合体细胞杂种克隆板,将新克隆的猪微卫星HAU02和HAU06定位于1q23-27、6q11-21.并对这两种基因定位方法的优缺点和应用进行了比较和讨论.这对于我国进行猪基因定位工作开展提供了思路;同时,新微卫星的定位为建立猪染色体物理图谱积累了有益的资料.     

Rapid detection of yeast rRNA genes with primed in situ (PRINS) labeling

In yeast, rRNA genes can be detected with the FISH technique using rRNA gene probes. This technique yields reliable, reproducible and precise results, but is time-consuming. Here, the primed in situ DNA synthesis (PRINS) procedure has been optimized for rapid detection of yeast rRNA genes. PRINS, which is as sensitive as PCR and allows cytological localization of analyzed sequences, can be adapted for various screening tests requiring fast labeling of rRNA genes.     

The polypeptide components of the parasporal fibres of Pasteuria penetrans

A variety of treatments were tested for their ability to solubilize the parasporal fibres from Pasteuria penetrans, a parasite of some plant–parasitic nematodes. Selective solubilization of the parasporal fibres resulted from some of the extraction procedures tested. Subsequent acrylamide gel electrophoresis and Western blotting of the resolved polypeptides, using polyclonal sera against the spores, disclosed up to 15 distinct bands, ranging in size from 12 to 195 kDa. An N-terminal amino acid sequence was obtained from a 50 kDa polypeptide and an oligonucleotide primer deduced from it. A whole cell, fluorescent, primed in situ labelling (PRINS) technique was adapted to be applicable to spores of P. penetrans and P. ramosa, a parasite of water fleas. Positive responses were obtained using the parasporal fibre primer on spores of the former but not of the latter organism, implying that this 50 kDa polypeptide is produced by P. penetrans but not by P. ramosa.