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.

Development of a fungal transformation system based on selection of sequences with promoter activity.

Abstract: A novel strategy was used to develop a transformation system for the plant pathogenic fungus Cochliobolus heterostrophus. Sequences capable of driving the expression of a gene conferring resistance to the antibiotic hygromycin B in C. heterostrophus were selected from a library of genomic DNA fragments and used, with the selectable marker, as the basis for transformation. The library of random 0.5- to 2.0-kilobase-pair fragments of C. heterostrophus genomic DNA was inserted at the 5' end of a truncated, promoterless Escherichia coli hygromycin B phosphotransferase gene (hygB) whose product confers resistance to hygromycin B. C. heterostrophus protoplasts were transformed with the library and selected for resistance. Resistant colonies arose at low frequency. Each colony contained a transformation vector stably integrated into chromosomal DNA. When the transforming DNA was recovered from the genome and introduced into C. heterostrophus, resistant colonies appeared at higher frequency. We determined the sequences of two of the C. heterostrophus DNA fragments which had been inserted at the 5' end of hygB in the promoter library and found that both made translational fusions with hygB. One of the two fusions apparently adds 65 and the other at least 86 amino acids to the N-terminus of the hygB product. Plasmids containing hygB-C. heterostrophus promoter fusions can be used unaltered to drive hygB expression in several other filamentous ascomycetes. This approach to achieving transformation may have general utility, especially for organisms with relatively undeveloped genetics.

Characterization and immunological identification of cDNA clones encoding two human DNA topoisomerase II isozymes

Abstract: Several DNA topoisomerase II (Topo II; EC 5.99.1.3) partial cDNA clones obtained from a human Raji-HN2 cDNA library were sequenced and two classes of nucleotide sequences were found. One member of the first class, SP1, was identical to an internal fragment of human HeLa cell Topo II cDNA described earlier. A member of the second class, SP11, shared extensive nucleotide (75%) and predicted peptide (92%) sequence similarities with the first two-thirds of HeLa Topo II. Each class of cDNAs hybridized to unique, nonoverlapping restriction enzyme fragments of genomic DNA from several human cell lines. Synthetic 24-mer oligonucleotide probes specific for each cDNA class hybridized to 6.5-kilobase mRNAs; furthermore, hybridization of probe specific for one class was not blocked by probe specific for the other. Antibodies raised against a synthetic SP1-encoded dodecapeptide specifically recognized the 170-kDa form of Topo II, while antibodies raised against the corresponding SP11-encoded dodecapeptide, or a second unique SP11-encoded tridecapeptide, selectively recognized the 180-kDa form of Topo II. These data provide genetic and immunochemical evidence for two Topo II isozymes.

AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression

Abstract: Based on homologies between the yeast DMC1 and the lily LIM15 meiosis-specific genes, degenerate PCR primers were designed that amplified the Arabidopsis DMC1 gene (AtDMC1). AtDMC1 genomic DNA (8 kb) was sequenced, and the transcript was characterized by reverse transcriptase-polymerase chain reaction (RT-PCR) and by 5' and 3' RACE (rapid amplification of cDNA ends). The AtDMC1 gene contains 15 exons and 14 introns. RNA in situ hybridization analysis showed that expression of the AtDMC1 is restricted to pollen mother cells in anthers and to megaspore mother cells in ovules. The AtDMC1 promoter was fused to the GUS reporter gene, and conferred meiosis-associated expression in both male and female floral lineages. Comparison of AtDMC1 isolated from Landsberg erecta ecotype to its Columbia allele ArLIM15, revealed the presence of a 1874 bp transposon-like element within the promoter region of ArLIM15. RT-PCR analysis showed that the expression levels of AtDMC1 and ArLIM15 are similar. Possible uses for the AtDMC1 promoter are discussed.

Molecular genetic basis of the human Rhesus blood group system.

Abstract: The Rhesus (RH) blood group locus is composed of two related structural genes, D and CcEe, that encode red cell membrane proteins carrying the D, Cc and Ee antigens. As demonstrated previously, the RhD–positive/RhD–negative polymorphism is associated with the presence or the absence of the D gene. Sequence analysis of transcripts and genomic DNA from individuals that belong to different Rh phenotypes were performed to determine the molecular basis of the C/c and E/e polymorphisms. The E and e alleles differ by a single nucleotide resulting in a Pro226Ala substitution, whereas the C and c alleles differ by six nucleotides producing four amino acid substitutions Cys16Trp, He60Leu, Ser68Asn and Ser103Pro. With the recent cloning of the RhD gene, these findings provide the molecular genetic basis that determine D, C, c, E and e specificities.