Genetic analysis, techniques and applications最新文献

The phylogenetic relationships between Bombycidae (Bombyx mori and Bombyx mandarina) and Saturniidae (Antheraea yamamai and Antheraea pernyi) were investigated based on large and small mitochondiral rRNA genes. About 430 bp of four kinds of PCR-amplified fragments were sequenced and aligned. For the 16S rRNA gene, B. mori shared a 98, 87 and 86% sequence homology with B. mandarina, A. yamamai and A. pernyi, and for the 12S rRNA gene, B. mori shared a 99, 89 and 88% sequence homology with B. mandarina, A. yamamai and A. pernyi, respectively. DNA sequence data were also used for a phylogenetic analysis. All of the trees showed monophyly for both Bombycidae and Saturniidae. The monophyly confidence limits of these trees were estimated using bootstrapping tests and measured more than 99% for all trees for both Bombycidae and Saturniidae.

Transcriptional regulation of Escherichia coli ATCC11105 penicillin amidase gene (pac) by cAMP receptor protein (CRP) and phenylacetic acid (PAA) was studied by using operon fusions with divergent reporter gene (lacZ, and phoA) constructs. A 150 bp DNA segment essential for the regulation of pac gene transcription by CRP and PAA was defined.

A procedure to produce an exact chromosomal replica of an insertion or insertion–deletion mutation produced in vitro in a plasmid with a ColE 1 origin of replication is presented. This procedure uses a previously described property of recD mutations (Biek DP, Cohen SN. J. Bacteriol. 1986;167:594–603) and is limited by (1) the compatibility of the new mutation with recD; and (2) the presence of some Escherichia coli DNA flanking the mutation.

The identification of the entire complement of genes expressed in a cell, tissue, or organism provides a framework for understanding biological properties and establishes a tool set for subsequent functional studies. The large-scale sequencing of randomly selected clones from cDNA libraries has been successfully employed as a method for identifying a large fraction of these expressed genes. However, this approach is limited by the inherent redundancy of cellular transcripts reflecting widely variant levels of gene transcription. As a result, a high percentage of transcript duplications are encountered as the number of sequenced clones accrues. To address this problem, we have developed a negative hybridization selection method that employs the hybridization of complex cDNA probes to high-density arrays of cDNA clones and the subsequent selection of clones with a null or low hybridization signal. This approach was applied to a cDNA library constructed from normal human prostate tissue and resulted in the reduction of highly expressed prostate cDNAs from 6.8 to 0.57% with an overall decline in clone redundancy from 33 to 11%. The selected clones also reflected a more diverse cDNA population, with 89% of the clones representing distinctly different cDNAs compared with 67% of the randomly selected clones. This method compares favorably with cDNA library re-association normalization approaches and offers several distinct advantages, including the flexibility to use previously prepared libraries, and the ability to employ an iterative screening approach for continued accrual of cDNAs representing rare transcripts.

Southern blotting, PCR, DGGE and DNA sequencing were used to study gene mutations in 52 unrelated Chinese Hemophilia A patients. 18 out of 34 severe cases had intron 22 inversions, 13 had small gene lesions, of which five are novel.

We report the screening of thirty-one YACs with a number of markers using polymerase chain reaction (PCR) to construct a physical map of part of human chromosome 17q21.3-q22. A contig of YACs covering about 4 Mb was constructed around the TCF11 gene at 68 cM from the most telomeric marker on the p arm, localizing TCF11 telomeric to genetic marker D17S1827. Both human and mouse P1-derived artificial chromosomes (PACs) containing TCF11 were isolated and characterized. The human heterochromatin protein 1 gene, HP1^Hsß, and its homologue in mouse, MoMOD1, were identified centromeric to TCF11.

We report a reliable method for PCR (polymerase chain reaction) amplification of genomic DNA from PET. This method uses DNA extraction with the QIAquick kit and amplification with AmpliTaq Gold. Amplification of up to 959 bp from PET was achieved with this combination which exceeds the current reported upper limit of 800 bp. In summary, the gradual activation of the AmpliTaq Gold during thermal cycling allows both for higher-fidelity and higher-throughput PCR amplification from PET. The use of the QIAquick kit for DNA purification of PET is sensitive, reproducible and suitable for management of a high number of samples.

Growth manipulation in fish is one of the targets of gene transfer experiments. The aim is to produce strains with improved growth performance. The transfer of growth hormone transgenes has been successful in many fish species. Now detailed knowledge of the molecular events that control growth in fish is necessary in order to efficiently manipulate this process. We have selected tilapia for our studies because these species are suitable for basic research as well as for the development of improved strains for aquaculture. Here we review the results of basic and applied research in the field of growth control and manipulation in tilapia. Our experiments produced new scientific results on growth control in tilapia. These results were used to develop a new aquacultured line with improved growth performance. Many of these results are probably applicable to other teleosts.