genomic DNA

About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.

Gene transfer, expression and inheritance of PRSV-rainbow trout-GH cDNA in the common carp, Cyprinus carpio (Linnaeus)

Abstract: A recombinant plasmid containing the Rous sarcoma virus-long terminal repeat (RSV-LTR) promoter linked to rainbow trout (Salmo gairdneri) growth hormone (GH) cDNA was microinjected into fertilized carp eggs. Genomic DNA extracted from pectoral fin of individual presumptive transgenic fish was analyzed by dot blot and Southern blot hybridization, using the RSV-LTR and/or the GH cDNA sequences as probes. Out of 365 presumptive transgenic fish analyzed, 20 individuals were found to contain pRSV-rtGH-cDNA sequence in the genomic DNA. Expression of the trout GH polypeptide was detected by immunobinding assay in the red blood cells of nine transgenic fish tested. The level of expression, however, varied among the transgenics and could not be correlated with exogenous DNA copy number. Although there was considerable variation in the sizes of the transgenic fish, those microinjected during the one-cell stage were (P less than 0.05) 22% larger, on the average, than their sibling controls. A randomly selected fraction of the progeny derived from crosses between transgenic males and non-transgenic females inherited the foreign DNA. These transgenic progeny grew faster (P less than 0.05) than their non-transgenic siblings.

Development of a species-diagnostic polymerase chain reaction assay for the identification of Culex vectors of St. Louis encephalitis virus based on interspecies sequence variation in ribosomal DNA spacers.

Abstract: Culex pipiens complex mosquitoes (Cx. p. pipiens and Cx. p. quinquefasciatus) are among the principal vectors of St. Louis encephalitis (SLE) virus in the eastern United States; Cx. restuans and Cx. salinarius play secondary roles in the transmission and maintenance of the virus cycle. Accurate identification of these three species in field collections is required for epidemiologic studies of SLE virus transmission. We have developed a polymerase chain reaction (PCR) assay for this purpose. Species-specific PCR primers were designed based on interspecies nucleic acid sequence variation in the first and second internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA gene array; however, insufficient variation was detected to differentiate between subspecies of the Cx. pipiens complex. The primers were used together in a single amplification reaction to correctly identify specimens to species using genomic DNA extracted from whole individual mosquitoes, DNA from triturated mosquito pools, or crude DNA from mosquito heads or legs.

Identification of self-incompatibility genotypes of almond by allele-specific pcr analysis

Abstract: In almond, gametophytic self-incompatibility is controlled by a single multiallelic locus (S-locus). In styles, the products of S-alleles are ribonucleases, the S-RNases. Cultivated almond in California have four predominant S-alleles (S a, S b, S c, S d). We previously reported the cDNA cloning of three of these alleles, namely S b, S c and S d. In this paper we report the cloning and DNA sequence analysis of the S a allele. The Sa-RNase displays approximately 55% similarity at the amino-acid level with other almond S-RNases (Sb, Sc, and Sd) and this similarity was lower than that observed among the Sb, Sc and Sd-RNases. Using the cDNA sequence, a PCR-based identification system using genomic DNA was developed for each of the S-RNase alleles. Five almond cultivars with known self-incompatibility (SI) geno-types were analyzed. Common sequences among four S-alleles were used to create four primers, which, when used as sets, amplify DNA bands of unique size that corresponded to each of the four almond S-alleles; S a (602 bp), S b (1083 bp), S c (221 bp) and S d (343 bp). All PCR products obtained from genomic DNA isolated from the five almond cultivars were cloned and their DNA sequence obtained. The nucleotide sequence of these genomic DNA fragments matched the corresponding S-allele cDNA sequence in every case. The amplified products obtained for the S a- and S b-alleles were both longer than that expected for the coding region, revealing the presence of an intron of 84 bp in the S a-allele and 556 bp in the S b-allele. Both introns are present within the site of the hypervariable region common in S-RNases from the Rosaceae family and which may be important for S specificity. The exon portions of the genomic DNA sequences were completely consistent with the cDNA sequence of the corresponding S-allele. A useful application of these primers would be to identify the S-genotype of progeny in a breeding program, new varieties in an almond nursery, or new grower selections at the seedling stage.

Stable integration and functional expression of flounder growth hormone gene in transformed microalga, Chlorella ellipsoidea.

Abstract: Chlorella is an attractive organism for complex recombinant protein production because of its eukaryotic characteristics and low cost for large-scale culture. Protoplasts of C. ellipsoidea were transformed with a vector containing the flounder growth hormone gene (fGH) under the control of the cauliflower mosaic virus 35S promoter, and the phleomycin resistance Sh ble gene under the control of the Chlamydomonas RBCS2 gene promoter. The presence of introduced DNA was first determined by PCR amplification of both the fGH and Sh ble genes from genomic DNA isolated from transformants and fGH protein expression was detected by immunoblot analysis. Over 400 microg of fGH protein expression per one liter culture containing 1 x 10(8) cells/ml was estimated by ELISA. Stable integration of introduced DNA was confirmed by Southern blot analysis of genomic DNA digested with restriction enzymes. The introduced DNA and fGH expression were detected after seven successive transfers in media devoid of phleomycin, but stably remained in the presence of the antibiotic. Flounder fry fed on the transformed Chlorella revealed a 25% growth increase after 30 days of feeding.