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.

Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants

Abstract: Very high-throughput sequencing technologies need to be matched by high-throughput functional studies if we are to make full use of the current explosion in genome sequences. We have generated a very large bacterial mutant pool, consisting of an estimated 1.1 million transposon mutants and we have used genomic DNA from this mutant pool, and Illumina nucleotide sequencing to prime from the transposon and sequence into the adjacent target DNA. With this method, which we have called TraDIS (transposon directed insertion-site sequencing), we have been able to map 370,000 unique transposon insertion sites to the Salmonella enterica serovar Typhi chromosome. The unprecedented density and resolution of mapped insertion sites, an average of one every 13 base pairs, has allowed us to assay simultaneously every gene in the genome for essentiality and generate a genome-wide list of candidate essential genes. In addition, the semiquantitative nature of the assay allowed us to identify genes that are advantageous and those that are disadvantageous for growth under standard laboratory conditions. Comparison of the mutant pool following growth in the presence or absence of ox bile enabled every gene to be assayed for its contribution toward bile tolerance, a trait required of any enteric bacterium and for carriage of S. Typhi in the gall bladder. This screen validated our hypothesis that we can simultaneously assay every gene in the genome to identify niche-specific essential genes.

Identification of a nonhistone chromosomal protein associated with heterochromatin in Drosophila melanogaster and its gene.

Abstract: Monoclonal antibodies were prepared against a fraction of nuclear proteins of Drosophila melanogaster identified as tightly binding to DNA. Four of these antibodies were directed against a 19-kilodalton nuclear protein; immunofluorescence staining of the polytene chromosomes localized the antigen to the alpha, beta, and intercalary heterochromatic regions. Screening of a lambda gt11 cDNA expression library with one of the monoclonal antibodies identified a recombinant DNA phage clone that produced a fusion protein immunologically similar to the heterochromatin-associated protein. Polyclonal sera directed against the bacterial lacZ fusion protein recognized the same nuclear protein on Western blots. A full-length cDNA clone was isolated from a lambda gt10 library, and its DNA sequence was obtained. Analysis of the open reading frame revealed an 18,101-dalton protein encoded by this cDNA. Two overlapping genomic DNA clones were isolated from a Charon 4 library of D. melanogaster with the cDNA clone, and a restriction map was obtained. In situ hybridization with these probes indicated that the gene maps to a single chromosome location at 29A on the 2L chromosome. This general strategy should be effective for cloning the genes and identifying the genetic loci of chromosomal proteins which cannot be readily assayed by other means.

A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex.

Abstract: A 0.59 kilobase DNA fragment cloned from an rDNA cistron of the mosquito Anopheles gambiae can be used as a probe to differentiate between A. gambiae, A. arabiensis, and A. melas, three morphologically identical sibling species in the A. gambiae complex which otherwise can be reliably distinguished only by polytene chromosome banding patterns. Although all are important (and often sympatric) African malaria vectors, their relative roles in malaria transmission have thus far been difficult to assess. The probe, an EcoRI-SalI fragment from the 3' end of the 28S beta coding region of the cistron, is present in all three species, but the species differ uniquely with respect to the location of an EcoRI site in the nontranscribed spacer (NTS) downstream of the fragment. We have routinely used the probe to identify A. gambiae complex mosquitoes to species on the basis of genomic DNA extracted from individual air dried specimens. A single mosquito abdomen provides more than sufficient DNA for the assay, and neither eggs nor a bloodmeal in the abdomen interfere with DNA yield. Moreover, the DNA extraction procedure does not degrade the bloodmeal IgG, so the residual protein pellet can be used to identify the mosquito bloodmeal source. Since the rDNA cistron organization as detected by the probe does not differ between male and female mosquitoes, the probe can be used for either sex. Preliminary experiments show that the probe is equally useful for mosquito larvae and pupae.

Structure and expression of the human cystatin C gene.

Abstract: The structural organization of the gene for the human cysteine-proteinase inhibitor cystatin C was studied. Restriction-endonuclease digests of human genomic DNA hybridized with human cystatin C cDNA and genomic probes produced patterns consistent with a single cystatin C gene and, also, the presence of six closely related sequences in the human genome. A 30 kb restriction map covering the genomic region of the cystatin C gene was constructed. The positions of three polymorphic restriction sites, found at examination of digests of genomic DNA from 79 subjects, were localized in the flanking regions of the gene. The gene was cloned and the nucleotide sequence of a 7.3 kb genomic segment was determined, containing the three exons of the cystatin C structural gene as well as 1.0 kb of 5'-flanking and 2.0 kb of 3'-flanking sequences. Northern-blot experiments revealed that the cystatin C gene is expressed in every human tissue examined, including kidney, liver, pancreas, intestine, stomach, antrum, lung and placenta. The highest cystatin C expression was seen in seminal vesicles. The apparently non-tissue-specific expression of this cysteine-proteinase inhibitor gene is discussed with respect to the structure of its 5'-flanking region, which shares several features with those of housekeeping genes.

Highly repeated sequences in mammalian genomes.

Abstract: Publisher Summary This chapter discusses the structure and organization of mammalian, highly repeated sequences at the molecular level. There is a description of tandemly repeated sequences, that are, satellites, and the segments that are interspersed among other genomic DNA sequences. The methods for the analysis of repeated DNA sequences are measurement of DNA renaturation kinetics and isopycnic centrifugation in gradients of CsCl and CsSO4. Eukaryote genomes can be divided into classes of DNA sequences according to the reiteration frequency. Many highly repeated sequences are in long tandem arrays. Some repetitive sequences are dispersed throughout major portions of genomes amid either other repeated sequences or sequences present only once per genome, that are, uniquesequences. The characteristic organizational feature of satellites and cryptic satellites is the tandem repetition of a unit DNA sequence. Satellite arrays resist separation by isopycnic centrifugation and instead remain within the main density fraction of genomic DNA. The repeat unit and its tandem organization can be revealed by restriction endonuclease digestion.