Biomedical subjects

J W Golden

Publications and source records attributed to J W Golden.

31 records · Page 2Linked to original sources

PubMed1990

Expression of the Anabaena sp. strain PCC 7120 xisA gene from a heterologous promoter results in excision of the nifD element.

An 11-kilobase-pair element interrupts the nifD gene in vegetative cells of Anabaena sp. strain PCC 7120. The nifD element normally excises only from the chromosomes of cells that differentiate into nitrogen-fixing heterocysts. The xisA gene contained within the element is required for the excision. Shuttle vectors containing the Escherichia coli tac consensus promoter fused to various 5' deletions of the xisA gene were constructed and conjugated into Anabaena sp. strain PCC 7120 cells. Some of the expression plasmids resulted in excision of the nifD element in a high proportion of vegetative cells. Excision of the element required deletion of an xisA 5' regulatory region which presumably blocks expression in Anabaena sp. strain PCC 7120 vegetative cells but not in E. coli. Strains lacking the nifD element grew normally in medium containing a source of combined nitrogen and showed normal growth and heterocyst development in medium lacking combined nitrogen. The xisA gene was shown to be the only Anabaena gene required for the proper rearrangement in E. coli of a plasmid containing the borders of the nifD element.

Blotting, Southern↗

PubMed1990

A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region.

A DNA-binding factor (VF1) partially purified from Anabaena sp. strain PCC 7120 vegetative cell extracts by heparin-Sepharose chromatography was found to have affinity for the xisA upstream region. The xisA gene is required for excision of an 11-kilobase element from the nifD gene during heterocyst differentiation. Previous studies of the xisA upstream sequences demonstrated that deletion of this region is required for the expression of xisA from heterologous promoters in vegetative cells. Mobility shift assays with a labeled 250-base-pair fragment containing the binding sites revealed three distinct DNA-protein complexes. Competition experiments showed that VF1 also bound to the upstream sequences of the rbcL and glnA genes, but the rbcL and glnA fragments showed only single complexes in mobility shift assays. The upstream region of the nifH gene formed a weak complex with VF1. DNase footprinting and deletion analysis of the xisA binding site mapped the binding to a 66-base-pair region containing three repeats of the consensus recognition sequence ACATT.

Base Sequence↗

PubMed1989

Excision of an 11-kilobase-pair DNA element from within the nifD gene in anabaena variabilis heterocysts.

The 3' region of the Anabaena variabilis nifD gene contains an 11-kilobase-pair element which is excised from the chromosome during heterocyst differentiation. We have sequenced the recombination sites which border the element in vegetative cells and the rearranged heterocyst sequences. In vegetative cells, the element was flanked by 11-base-pair direct repeats which were identical to the repeats present at the ends of the nifD element in Anabaena sp. strain PCC 7120 (Anabaena strain 7120). Although Anabaena strain 7120 and A. variabilis are quite distinct in many ways, the overall sequence similarity between the two strains for the regions sequenced was 96%. Like the Anabaena strain 7120 element, the A. variabilis element was excised in heterocysts to produce a functional nifD gene and a free circularized element which was neither amplified nor degraded. The Anabaena strain 7120 xisA gene is located at the nifK-proximal end of the nifD element and is required for excision of the element in heterocysts. The A. variabilis element also contained an xisA gene which could complement a defective Anabaena strain 7120 xisA gene. A. variabilis did not contain the equivalent of the Anabaena strain 7120 fdxN 55-kilobase-pair element.

Amino Acid Sequence↗

PubMed1988

Genome rearrangement and nitrogen fixation in Anabaena blocked by inactivation of xisA gene.

Two genome rearrangements involving 11- and 55-kilobase DNA elements occur during the terminal differentiation of an Anabaena photosynthetic vegetative cell into a nitrogen-fixing heterocyst. The xisA gene, located on the nifD 11-kilobase DNA element, was inactivated by recombination between the chromosome and a copy of the xisA gene that was mutated by inserting an antibiotic gene cassette. Site-directed inactivation of the Anabaena xisA gene blocked rearrangement of the 11-kilobase element and nitrogen fixation, but did not affect rearrangement of the 55-kilobase element, heterocyst differentiation, or heterocyst pattern formation.

Cyanobacteria↗

PubMed1988

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.

Chromosome Deletion↗

PubMed1986

Identification and sequence of a gene required for a developmentally regulated DNA excision in Anabaena.

Vegetative cells of the cyanobacterium Anabaena contain an 11 kb DNA element within the coding region of the nifD gene. This element is excised by site-specific recombination between directly repeated 11 bp sequences at each of its ends during differentiation of nitrogen-fixing cells called heterocysts. Site-specific recombination, leading to the same rejoined nifD gene, was observed during propagation in E. coli of a fragment containing the 11 kb element and flanking sequences. An assay for excision of the element in E. coli was developed, based on mini-Mu-lac transposition into the element. Since the 11 kb element lacks an origin of replication, its excision results in loss of lac and conversion of blue colony-forming cells to white on X-gal plates. Insertion and deletion mutagenesis identified a region of the element needed for excision. Mutations in this region could be complemented by a 6 kb fragment containing an open reading frame that runs counter to those of the nif genes, beginning 240 bp from the recombination site.

Base Sequence↗

PubMed1985

A gene affecting production of the Caenorhabditis elegans dauer-inducing pheromone.

A nematode mutant lacking pheromone activity does not enter the developmentally arrested dispersal stage called the dauer larva unless exogenous pheromone is added to the growth medium, indicating that the pheromone is required for wild-type dauer larva formation. In contrast, a class of temperature-sensitive mutant forms dauer larvae even in the absence of detectable pheromone, indicating that such mutants bypass the normal pheromone requirement. A rapid bioassay of pheromone produced by individual nematodes has been developed for genetic analysis of pheromone production.

Animals↗

PubMed1984

The Caenorhabditis elegans dauer larva: developmental effects of pheromone, food, and temperature.

Three environmental cues influence both the entry into and exit from the developmentally arrested dispersal stage called the dauer larva: a dauer-inducing pheromone, food, and temperature. The pheromone, which is a measure of population density, induces dauer larva formation at the second (L2) molt and inhibits recovery in a dose-dependent manner. Food acts competitively to reduce the frequency of dauer larva formation and to enhance recovery. The pheromone causes a specific extension of the second larval stage, coupled with a transient decrease in the growth rate of the L2. Second-stage larvae grown in the presence of added pheromone are morphologically distinguishable from L2 larvae grown without pheromone. We have named the pre-dauer L2 larva the L2d. Commitment to dauer larva formation can occur at the L2d molt. When L2d larvae are shifted out of pheromone to a lawn of E. coli just before the L2d molt, a few worms complete development into dauer larvae. In contrast, worms are essentially committed to the non-dauer life cycle by the first larval molt if the L1 larvae are not grown in appropriately high levels of pheromone. In the presence of pheromone, the percentage of dauer larva formation is enhanced at higher temperatures within the normal growth range. Temperature down-shifts induce dauer larva recovery. Temperature-shift experiments show that the enhancement of dauer larva formation requires exposure to the higher temperature around the L1 molt. Two sensory mutants defective in thermotaxis are altered in their sensitivity to the dauer-inducing pheromone, but their pheromone response retains temperature dependence. Response of dauer larvae to environmental cues is highly age dependent, with older dauer larvae exhibiting an increased tendency to recover.

Animals↗

PubMed1984

A pheromone-induced developmental switch in Caenorhabditis elegans: Temperature-sensitive mutants reveal a wild-type temperature-dependent process.

Formation of a developmentally arrested dispersal stage called the dauer larva is enhanced by a Caenorhabditis-specific pheromone and is inhibited by increasing amounts of food. Pheromone-induced dauer larva formation of three tested wild-type strains is temperature-dependent, so that an increased percentage of the population forms dauer larvae at 25 degrees C compared to lower temperatures. Dauer-defective mutants fail to respond to added pheromone, and some behavioral mutants affected in thermotaxis or egg-laying also exhibit abnormal responses. Temperature-sensitive (ts) dauer-constitutive mutants form dauer larvae at a restrictive temperature regardless of environmental stimuli. At the permissive temperature (17.5 degrees C), alleles of six out of seven dauer-constitutive genes tested overrespond to the dauer-inducing pheromone. All known mutations in daf-4 (eight alleles) and daf-7 (five alleles) produce a ts dauer-constitutive phenotype. One daf-4 and one daf-7 allele are suppressed by the amber nonsense suppressor, sup-7(st5). At least these two dauer-constitutive mutations are likely to cause production of nonfunctional rather than ts gene products. These mutations appear to indirectly result in a ts phenotype by enhancing the expression of a wild-type ts developmental process.

Animals↗

PubMed1982

A pheromone influences larval development in the nematode Caenorhabditis elegans.

A Caenorhabditis-specific pheromone and the food supply influence both entry into and exit from a developmentally arrested juvenile stage called the dauer larva. The pheromone increases the frequency of dauer larva formation and inhibits recovery but does not affect adult behavior such as chemotaxis and egg laying. The fatty acid--like pheromone has been partially purified and characterized by a new bioassay. If similar developmental control mechanisms are used by parasitic nematodes, such mechanisms might be exploited to develop highly selective anthelmintic agents.

Animals↗

PubMed1980

A new filamentous phage cloning vector: fd-tet.

We have constructed a hybrid chromosome composed of the genome of wild-type fd (a filamentous, male-specific bacteriophage) and a segment of transposon Tn10 coding for tetracycline resistance but not including the Tn10 insertion sequences. The hybrid phage infects male E. coli, thereby transducing the infected cells to tetracycline resistance. The phage DNA can also be propagated in F- cells after transfection. This new phage, fd-tet, may be used as a cloning vector to produce large quantities of cloned DNA in single-stranded form. Its usefulness has been demonstrated by cloning of a fragment from bacteriophage lambda. Some unexpected sequence alterations have been identified in lambda cloning experiments.

Coliphages↗

PubMedDate not supplied

Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena.

Nitrogen fixation by the cyanobacterium Anabaena is carried out in heterocysts, specialized, non-dividing cells which differentiate under conditions of ammonia or nitrate deprivation. In Anabaena, heterocyst differentiation is accompanied by rearrangement of some nitrogen fixation genes. A site-specific recombination between an 11 base-pair direct repeat sequence flanking the nifK and nifD genes removes 11 kilobases of intervening DNA, resulting in juxtaposition of the two genes and an alteration of the nifD protein-coding sequence.

Cell Differentiation↗

PubMedDate not supplied

Different recombination site specificity of two developmentally regulated genome rearrangements.

In the absence of a combined nitrogen source, such as ammonia, approximately every tenth vegetative cell along filaments of the cyanobacterium Anabaena develops into a heterocyst, a terminally differentiated cell that is morphologically and biochemically specialized for nitrogen fixation. At least two specific DNA rearrangements involving the nitrogen-fixation (nif) genes occur during heterocyst differentiation, one within the nifD gene and the other near the nifS gene. The two rearrangements have several properties in common. Both occur quantitatively in all heterocyst genomes, both occur at approximately the same developmental time, late in the process of heterocyst differentiation, and both result from site-specific recombination between short repeated DNA sequences. We report here the nucleotide sequences found at the site of recombination near the nifS gene. These sequences differ from those found previously for the nifD rearrangement, suggesting that the two rearrangements are catalysed by different enzymes and may be regulated independently. We also show that the nifS gene is transcribed only from rearranged genomes.

Cyanobacteria↗