Rapid and reliable fluorescent cycle sequencing of double-stranded templates

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects

Cloning, localization, and permanent expression of a Drosophila octopamine receptor

A cDNA for a member of the G protein-coupled receptor family was isolated from Drosophila using a probe derived from a human β2-adrenergic receptor cDNA. This Drosophila receptor gene is localized at 99A10-B1 on the right arm of chromosome 3 and is preferentially expressed in Drosophila heads. The insect octopamine receptor has been permanently expressed in mammalian cells, where it mediates the attenuation of adenylate cyclase activity and exhibits a pharmacological profile consistent with an octopamine type 1 receptor. Sequence and pharmacological comparisons indicate that the octopamine receptor is unique but closely related to mammalian adrenergic receptors, perhaps as an evolutionary precursor

An adipose tissue-specific β-adrenergic receptor. Molecular cloning and down-regulation in obesity

Clones encoding an atypical β-adrenergic receptor were isolated from a rat brown adipose tissue cDNA library. This receptor expressed in Chinese hamster ovary (CHO) cells displays a low affinity for β-adrenergic antagonists and a high affinity for BRL 37344, an agonist that selectively stimulates lipolysis in adipose tissue. The rank order of potency for agonist-mediated increases in intracellular cAMP in transfected cells correlates with that for agonist-mediated stimulation of lipolysis in brown adipocytes. Northern blot analysis demonstrates that this receptor subtype is expressed only in brown and white adipose tissue where it represents the predominant β-receptor subtype. The amount of atypical β-adrenergic receptor present in adipose tissue of obese (fa/fa) Zucker rats is reduced by up to 71% as compared with lean (Fa/Fa) control animals. These findings suggest that a change in the expression of this β-adrenergic receptor subtype may play a role in obesity

Sequencing and analysis of genomic fragments from the NF1 locus

The sequence of five non-contiguous genomic fragments encompassing 14.4 kilobases from the NF1 locus have been determined by fluorescence-based automated DNA sequence analysis. These fragments included one kilobase of the NF1 coding region, which resulted in the identification of the intron/exon boundaries of five exons. Based on these sequences, five new NF1 exon-PCR assays have been developed, that could be useful for detecting new NF1 mutations. The genomic sequences were analyzed for the presence of Alu repetitive elements and their classification is described. This analysis may provide some insight into the characterization of genetic rearrangements resulting in disruption of the NF1 gene

Cloning, sequence analysis and chromosome localization of a Drosophila muscarinic acetylcholine receptor

Two cDNA clones (3.7 kb and 4.8 kb) encoding a Drosophila muscarinic acetylcholine receptor were isolated from a Drosophila head cDNA library and characterized by automated DNA sequence analysis. The Drosophila muscarinic receptor contains 788 amino acids with a calculated Mr of 84 807 and displays greater than 60% homology with mammalian muscarinic receptors. The muscarinic receptor maps to the tip of the right arm of the second chromosome of the Drosophila genome. © 1989

Complementary DNA sequencing: Expressed sequence tags and human genome project

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields

Automated DNA sequencing and analysis of 106 kilobases from human chromosome 19q13.3

A total of 116,118 basepairs (bp) derived from three cosmids spanning the ERCC1 locus of human chromosome 19q13.3 have been sequenced with automated fluorescence-based sequencers and analysed by polymerase chain reaction amplification and computer methods. The assembled sequence forms two contigs totalling 105,831 bp, which contain a human fosB proto-oncogene, a gene encoding a protein phosphatase, two genes of unknown function and the previously-characterized ERCC1 DNA repair gene. This light band region has a high average density of 1.4 Alu repeats per kilobase. Human chromosome light bands could therefore contain up to 75,000 genes and 1.5 million Alu repeats