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W F Doolittle

Publications and source records attributed to W F Doolittle.

At least 19 recordsLinked to original sources
PubMed1992

Mevinolin-resistant mutations identify a promoter and the gene for a eukaryote-like 3-hydroxy-3-methylglutaryl-coenzyme A reductase in the archaebacterium Haloferax volcanii.

Both eukaryotes and archaebacteria use 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase to synthesize mevalonate, which eukaryotes employ in the production of sterols and archaebacteria need for the isoprenoid side chains of their unique and characteristic lipids. The drug mevinolin inhibits HMG-CoA reductase in eukaryotes and in the halophilic archaebacteria, and we have used a spontaneous mutation to mevinolin resistance in the construction of a selectable shuttle vector for Haloferax volcanii. Sequence analysis shows that this resistance determinant encodes an HMG-CoA reductase very like its eukaryotic homologs, but sharing with the one sequenced eubacterial HMG-CoA reductase (that of Pseudomonas mevalonii) few residues other than those common to all HMG-CoA reductases. Characterization of several spontaneous mevinolin-resistant mutants reveals that they are of two sorts: amplifications of the HMG-CoA reductase gene with varying amounts of flanking sequence, and point mutants upstream of the HMG-CoA reductase coding region. We compared sequence and expression of a mutant gene of the latter class to those of the wild-type gene. The point mutation found affects the TATA box-like "distal promoter element," results (like gene amplification) in resistance through the synthesis of excess gene product, and provides the first true genetic definition of an archaebacterial promoter.

Amino Acid Sequence↗
PubMed1992

Localizing genes on the map of the genome of Haloferax volcanii, one of the Archaea.

We have assigned genetic markers to locations on the physical map of the genome of the archaeon Haloferax volcanii, using both a physical method (hybridization) and a more specific genetic technique (transformation with cosmids). Hybridizations were against restriction digests of each of 151 cosmids making up a minimally overlapping set and covering 96% of the genome. Results with a cloned insertion sequence and a tRNA probe indicated that transposable elements are concentrated on two of the four plasmids of this species, whereas regions complementary to tRNA are largely chromosomal. For a genetic analysis of genes involved in the biosynthesis of amino acids, purines, and pyrimidines, we used cosmid transformation to assign 139 of 243 ethyl methanesulfonate-induced auxotrophic mutations, generated and characterized for this study, to single cosmids or pairs of cosmids from the minimal set. Mutations affecting the biosynthesis of uracil, adenine, guanine, and 14 amino acids have been mapped in this way. All mutations mapped to the 2920-kilobase-pair chromosome of Hf. volcanii and seemed uniformly distributed around this circular replicon. In some cases, many mutations affecting a single pathway map to the same or overlapping cosmids, as would be expected were genes for the pathway linked. For other biosynthetic pathways, several unlinked genetic loci can be identified.

Archaea↗
PubMed1992

Variable arrangement of 5S ribosomal genes within the ribosomal DNA repeats of arthropods.

The polymerase chain reaction and hybridization to genomic blots were used to investigate whether the previously observed inclusion of 5S ribosomal RNA genes in the 28S-18S ribosomal DNA intergenic regions of some crustacean species (copepods) could also be detected in other arthropods. Such an arrangement was found not only in other calanoid copepod species but also in a cirriped, an euphausid, and a spider. It is interesting that species from two different calanoid copepod genera do not have this type of arrangement. We conclude that the inclusion of 5S ribosomal RNA genes within the ribosomal DNA repeats has probably occurred repeatedly during the evolution of arthropod species and that the mechanism(s) responsible for these insertions could also be responsible for their loss.

Animals↗
PubMed1992

Transformation of a bop-hop-sop-I-sop-II-Halobacterium halobium mutant to bop+: effects of bacteriorhodopsin photoactivation on cellular proton fluxes and swimming behavior.

We have transformed Pho81, a Halobacterium halobium mutant strain which does not contain any of the four retinylidene proteins known in this species, with the bop gene cluster to create Pho81BR, a BR+HR-SR-I-SR-II-strain. The absorption spectrum, pigment reconstitution process, light-dark adaptation and photochemical reaction cycle of the expressed protein are indistinguishable from those of native bacteriorhodopsin (BR) in purple membrane of wild type strains. Strain Pho81BR permits for the first time characterization of effects of BR photoactivation alone on cell swimming behavior and energetics in the absence of the spectrally similar phototaxis receptor sensory rhodopsin I (SR-I) and electrogenic chloride pump halorhodopsin (HR). A non-adaptive upward shift in spontaneous swimming reversal frequency occurs following 3 s of continuous illumination of Pho81BR cells with green light (550 +/- 20 nm). This effect is abolished by low concentrations of the proton ionophore carbonylcyanide m-chlorophenylhydrazone. Although BR does not mediate phototaxis responses in energized Pho81BR cells under our culture conditions, proton pumping by BR in Pho81BR cells partially deenergized by inhibitors of respiration and adenosine triphosphate synthesis results in a small attractant response. Based on our measurements, we attribute the observed effects of BR photoactivation on swimming behavior to secondary consequences of electrogenic proton pumping on metabolic or signal transduction pathways, rather than to primary sensory signaling such as that mediated by SR-I. Proton extrusion by BR activates gated proton influx ports resulting in net proton uptake in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriorhodopsins↗
PubMed1992

Transformation of members of the genus Haloarcula with shuttle vectors based on Halobacterium halobium and Haloferax volcanii plasmid replicons.

We have stably transformed both Haloarcula vallismortis and Haloarcula hispanica with the halobacterium-Escherichia coli shuttle vectors pWL102 (based on the Haloferax volcanii pHV2 replicon) and pUBP2 (based on the Halobacterium halobium pHH1 replicon). Haloferax volcanii, Halobacterium halobium, and Haloarcula vailismortis are equally distant from one another and span the phylogenetic depth of the halophilic Archaea; thus, these vectors may be generally useful for the halophiles. Both Haloarcula vallismortis and Haloarcula hispanica exhibit previously unreported complex life cycles and are therefore significant as genetically approachable models of cellular differentiation within the Archaea.

Genetic Vectors↗
PubMed1991

Detailed physical map and set of overlapping clones covering the genome of the archaebacterium Haloferax volcanii DS2.

An integrated approach of "bottom up" and "top down" mapping has produced a minimal set of overlapping cosmid clones covering 96% of the 4140 kilobase-pairs (kbp) Haloferax volcanii DS2 genome and a completely closed physical map. This genome is partitioned into five replicons: a 2920 kbp chromosome and four plasmids, of 690 kbp (pHV4), 442 kbp (pHV3), 86 kbp(pHV1) and 6.4 kbp (pHV2). A restriction map for six infrequently-cutting restriction enzymes was constructed, representing a total of 903 sites in the cloned DNA. We have placed the two ribosomal RNA operons, the genes for 7 S RNA and for RNaseP RNA and 22 protein-coding genes on the map. Restriction site frequencies show significant variation in different portions of the genome. The regions of high site density correspond to halobacterial satellite or FII DNA which includes two small regions of the chromosome, the plasmids pHV1 and pHV2, and half of pHV4, but not pHV3.

Archaea↗
PubMed1990

Genes for tryptophan biosynthesis in the archaebacterium Haloferax volcanii.

Recent technical advances permit direct genetic approaches for isolating genes and mapping auxotrophic mutations in the halophilic archaebacterium (Archaea) Haloferax volcanii. Twenty-nine mutations in tryptophan biosynthesis mapped to two separate chromosomal locations. DNA sequencing of one gene cluster shows a unique gene order (trpCBA) and unusual potential secondary structures in the 5'-flanking region.

Alkadienes↗
PubMed1990

Characterization of a gene involved in histidine biosynthesis in Halobacterium (Haloferax) volcanii: isolation and rapid mapping by transformation of an auxotroph with cosmid DNA.

Techniques for the transformation of halophilic archaebacteria have been developed recently and hold much promise for the characterization of these organisms at the molecular level. In order to understand genome organization and gene regulation in halobacteria, we have begun the characterization of genes involved in amino acid biosynthesis in Halobacterium (Haloferax) volcanii. These studies are facilitated by the many auxotrophic mutants of H. volcanii that have been isolated. In this project we demonstrate that cosmid DNA prepared from Escherichia coli can be used to transform an H. volcanii histidine auxotroph to prototrophy. A set of cosmid clones covering most of the genome of H. volcanii was used to isolate the gene which is defective in H. volcanii WR256. Subcloning identified a 1.6-kilobase region responsible for transformation. DNA sequence analysis of this region revealed an open reading frame encoding a putative protein 361 amino acids in length. A search of the DNA and protein data bases revealed that this open reading frame encodes histidinol-phosphate aminotransferase (EC 2.6.1.9), the sequence of which is also known for E. coli, Bacillus subtilis, and Saccharomyces cerevisiae.

Amino Acid Sequence↗
PubMed1989

Shuttle vectors for the archaebacterium Halobacterium volcanii.

Progress in archaebacterial molecular biology requires tools for genetic analysis. We describe vectors that can be selected and maintained in either Halobacterium volcanii or Escherichia coli. A genetic determinant for resistance to the 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor mevinolin was isolated by "shotgun cloning" into a derivative of the endogenous H. volcanii plasmid pHV2, to form pWL2, which transforms sensitive H. volcanii to mevinolin resistance at high frequency. The resistance determinant, portions of pHV2, and an ampicillin- and tetracycline-resistance-conferring pBR322 derivative, pAT153, were ligated together to form the shuttle vectors pWL101 and pWL102. We describe conditions for the use of these vectors and provide preliminary definition of regions essential for drug resistance and for plasmid replication and maintenance.

Chromosome Deletion↗
PubMed1989

Transformation of the archaebacterium Halobacterium volcanii with genomic DNA.

We describe optimization of a transformation system for the halophilic archaebacterium Halobacterium volcanii. Transformation of spheroplasts in the presence of polyethylene glycol permits the uptake and expression of high-molecular-weight linear fragments of genomic DNA as well as plasmid or bacteriophage DNA. Transformations can be performed with either fresh or frozen cell preparations. Auxotrophic mutants were transformed to prototrophy with genomic DNA from wild-type cells with efficiencies of 5 x 10(4)/micrograms of DNA and frequencies of 8 x 10(-5) per regenerated spheroplast. The overall efficiency of transformation with genomic DNA implies that genetic recombination is an efficient process in H. volcanii.

DNA, Bacterial↗
PubMed1989

Genome mapping in halobacteria.

The goal of our research is to produce an ordered set of cosmid clones for each of several species of halobacteria for use in physical and genetic mapping. These maps will answer questions about genome evolution and about gene organization and regulation in this archaebacterial lineage. Progress in cloning and mapping the genome of Halobacterium volcanii DS2 (synonym Haloferax volcanii DS2) is reported. Overlapping cosmids are recognized by a strategy which makes use of the distinctive restriction fragments around relatively rare restriction sites. Each site recognized by the infrequently cutting restriction enzymes is a landmark from which to identify different regions of the genome. The main advantage of this strategy is that only a small overlap (10-20%) between cosmid clones is required, resulting in a correspondingly small number of cosmid clones to be analyzed. The certainty of overlap is high, and computation is simple. The final 5-10% of each genome is cloned, linked, and identified by chromosome walking methods. Hybridization of cloned homologous or heterologous genes and of stable RNAs to the minimal cosmid set localizes these genes on the physical map. Additional genes have been and will be cloned by complementation of auxotrophic mutants, or as determinants of resistance to antibiotics.

Chromosome Mapping↗
PubMed1989

Transformation methods for halophilic archaebacteria.

We present a practical description of polyethylene glycol mediated spheroplast transformation of Halobacterium halobium and Halobacterium volcanii. This method has been applied to phage DNA transfection, plasmid DNA transformation, and transformation with linear fragments of high molecular weight genomic DNA. Efficient spheroplast regeneration allows uncomplicated recovery of transformed progeny. Transformations can be performed equally well using fresh or frozen cell preparations. These methods should find application in molecular cloning, genetic fine mapping, and strain construction.

Archaea↗
PubMed1988

Bacterial evolution.

The deliberate application of the methodology of contemporary molecular genetics to problems in bacterial systematics has led to a broad new understanding of the evolutionary history of both prokaryotes and eukaryotes. In this review, I discuss some of the major conclusions of this endeavour and try to predict future directions.

Animals↗
PubMed1987

Unusual ribosomal RNA gene organization in copepods of the genus Calanus.

Ribosomal RNA genes in the nuclear genomes of eukaryotes are generally found in tandemly repeated units encoding 18 S, 5.8 S and 28 S rRNA (in that order). 5 S rRNA genes typically lie outside these units, most often in tandem clusters coding exclusively for 5 S rRNA. Inclusion of 5 S genes within the 18 S-5.8 S-28 S repeat unit is known only for certain protozoa and fungi. Here we report that, in the copepod Calanus finmarchicus, single 5 S genes are included within many or all of the 18 S-5.8 S-28 S repeat units. Sequence analyses of regions cloned from two of these repeat units show that they indeed include 5 S genes (which are distal to 28 S genes) and that these are transcribed from opposite strands.

Animals↗