Using FTA® cards to store avian blood samples for genetic studies. Their application in sex determination

FTA® cards were used for long‐term storage of avian blood samples. Blood DNA was extracted by a simple method and used in PCR for sex identification of adult and nestling Great Grey Shrikes Lanius excubitor.     

DNA Sequences from Formalin-Fixed Nematodes: Integrating Molecular and Morphological Approaches to Taxonomy

To effectively integrate DNA sequence analysis and classical nematode taxonomy, we must be able to obtain DNA sequences from formalin-fixed specimens. Microdissected sections of nematodes were removed from specimens fixed in formalin, using standard protocols and without destroying morphological features. The fixed sections provided sufficient template for multiple polymerase chain reaction-based DNA sequence analyses.     

Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia

Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.     

How to Use Genetic Data to Distinguish Between Natural and Human-Mediated Introduction of <Emphasis Type="Italic">Littorina littorea</Emphasis> to North America

The rapid range southward expansion of the periwinkle Littorina littorea from the Canadian maritimes has fueled a long-running debate over whether this species was introduced to North America by human activity. A reappraisal of the mitochondrial DNA sequence evidence finds considerable endemic allelic diversity in the American population. The degree of endemic genetic diversity is higher than expected from human-mediated colonization, but not so much to suggest that it survived the last glacial maximum in America. Coalescent estimates of population divergence agree that colonization of America preceded European contact. A reappraisal of the ITS nuclear sequence data finds extensive recombination. Taking this recombination into account strengthens the genetic case against human-mediated introduction. Finally, a reappraisal of conflicting allozyme studies from the 1970’s supports a claim of limited divergence between American and European populations. This is consistent with post-glacial colonization, but the allozyme data cannot distinguish between natural or human-mediated colonization. Taken as a whole, the DNA sequence data supports the many sub-fossil reports of an American L. littorea population in the Canadian maritimes that preceded even the first visits by the Vikings.     

Strategies for signal amplification in nucleic acid detection

Many aspects of molecular genetics necessitate the detection of nucleic acid sequences. Current approaches involving target amplification (in situ PCR, Primed in situ Labeling, Self-Sustained Sequence Replication, Strand Displacement Amplification), probe amplification (Ligase Chain Reaction, Padlock Probes, Rolling Circle Amplification) and signal amplification (Tyramide Signal Amplification, Branched DNA Amplification) are summarized in the present review, together with their advantages and limitations.     

恶性疟原虫抗原表位的亚单位疫苗与DNA疫苗的联合免疫

构建了含有恶性疟原虫抗原基因 ( AWTE)的真核表达质粒 p CMV- AWTE,以及能在大肠杆菌中得到分泌性表达的原核表达质粒 p MC0 5 ,表达的蛋白 AWTE保持了疟原虫抗原的抗原性。将 p CMV- AWTE以及 AWTE两者混合各 1 0μg鼻腔免疫小鼠 ,一次后诱导机体产生了较高水平的体液免疫及细胞免疫     

Molecular data reveal convergence in fruit characters used in the classification of Thlaspi s. I. (Brassicaceae)

Phylogenetic relationships of 18 Thlaspi s.l. species were inferred from nuclear ribosomal internal transcribed spacer (ITS) sequence data. These species represent all sections of the basic classification system of Schulz primarily based on fruit characters. The molecular phylogeny supported six clades that are largely congruent with species groups recognized by Meyer on the basis of differences in seed coat anatomy, i.e. Thlaspi s. s., Thlaspkeras, Moccaea {Raparia included), Microthhspi, Vania and Neurotropy. Some of these lineages include species which are morphologically diverse in fruit shape (e.g. Thlaspi s. s.: T. arvense - fruits broadly winged, T. ceratocarpum - fruits with prominent horns at apex, T. alliaceum - fruits very narrowly winged). Furthermore, the same fruit shape type is distributed among different clades. For instance, fruits with prominent horns at apex are found in Thlaspi s. s. ( T. ceratocarpum) and Thlaspiceras (T oxyceras). These results clearly indicate convergence in fruit characters previously used for sectional classification in Thlaspi s. l.     

Curiously Modern DNA for a ``250 Million-Year-Old' Bacterium

Studies of ancient DNA have attracted considerable attention in scientific journals and the popular press. Several of the more extreme claims for ancient DNA have been questioned on biochemical grounds (i.e., DNA surviving longer than expected) and evolutionary grounds (i.e., nucleotide substitution patterns not matching theoretical expectations for ancient DNA). A recent letter to Nature from Vreeland et al. (2000), however, tops all others with respect to age and condition of the specimen. These researchers extracted and cultured a bacterium from an inclusion body from what they claim is a 250 million-year (Myr)-old salt crystal. If substantiated, this observation could fundamentally alter views about bacterial physiology, ecology and evolution. Here we report on molecular evolutionary analyses of the 16S rDNA from this specimen. We find that 2-9-3 differs from a modern halophile, Salibacillus marismortui, by just 3 unambiguous bp in 16S rDNA, versus the ∼59 bp that would be expected if these bacteria evolved at the same rate as other bacteria. We show, using a Poisson distribution, that unless it can be shown that S. marismortui evolves 5 to 10 times more slowly than other bacteria for which 16S rDNA substitution rates have been established, Vreeland et al.'s claim would be rejected at the 0.05 level. Also, a molecular clock test and a relative rates test fail to substantiate Vreeland et al.'s claim that strain 2-9-3 is a 250-Myr-old bacterium. The report of Vreeland et al. thus falls into a long series of suspect ancient DNA studies. Received: 12 April 2001 / Accepted: 9 June 2001     

DNA deletions in spontaneous chloramphenicol-sensitive mutants of Streptomyces coelicolor A 3(2) and Streptomyces lividans 66

Summary A mutant of Streptomyces coelicolor A3(2) highly resistant to chloramphenicol was selected. It had amplified some chromosomal DNA fragments to a copy number of 20–50. Some of the amplified fragments were cloned and used as hybridisation probes to investigate the spontaneous chloramphenicol-sensitive mutants which occur at high frequency in this species and the closely related species Streptomyces lividans 66. These investigations demonstrated that chloramphenicol sensitivity in both species is associated with large deletions that are at least 40 kb in length.