William J. Brammar

A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes

Lambda derivatives are described that can be used for cloning DNA fragments of about 20 kilobase pairs (kb) generated by restriction enzymes EcoRi, HindIII, BamHI, MboI and BglII. Recombinants can be selected by their Spi- phenotype and their propagation is facilitated by the presence of a chi site.

A single aspartate residue is involved in both intrinsic gating and blockage by Mg2+ of the inward rectifier, IRK1.

1. We describe the effects on channel function of changing an aspartate residue (Asp172) in a membrane‐spanning alpha‐helix of the murine inward rectifier, IRK1, by site‐directed mutagenesis. 2. Alteration of Asp172 to Glu (charged) or to Gln or Asn (polar but uncharged) produced functional channels showing inward rectification, though rectification was weaker with Gln and Asn. 3. Intrinsic gating around the potassium equilibrium potential, EK, was conserved only if the charge on residue 172 was conserved. Currents through channels with Gln or Asn in this position showed no time dependence under hyperpolarization. 4. The change from Asp to Gln also reduced the affinity for internal Mg2+ at least fivefold, indicating that Asp172 also forms part of the site for Mg2+ blockage. 5. The consequences for channel structure of Asp172 lining the pore are discussed.

The intrinsic gating of inward rectifier K+ channels expressed from the murine IRK1 gene depends on voltage, K+ and Mg2+

1. We describe the cloning of the inward rectifier K+ channel IRK1 from genomic DNA of mouse; the gene is intronless. 2. The IRK1 gene can be stably expressed in murine erythroleukaemia (MEL) cells. Such transfected cells show inward rectification under whole‐cell recording. 3. Channels encoded by the IRK1 gene have an intrinsic gating that depends on voltage and [K+]o. Rate constants are reduced e‐fold as the driving force on K+(V‐EK) is reduced by 24.1 mV. 4. Removal of intracellular Mg2+ permits brief outward currents under depolarization. The instantaneous current‐voltage relation may be fitted by an appropriate constant field expression. 5. Removal of intracellular Mg2+ speeds channel closure at positive voltages. In nominally zero [Mg2+]i, rate constants for the opening and closing of channels, processes which are first order, are similar to those of native channels.

The ontogeny of peroxisome-proliferator-activated receptor gene expression in the mouse and rat

The expression of the gene coding for peroxisome-proliferator-activated receptor (PPAR), a novel transacting factor belonging to the steroid superfamily, has been determined in the mouse and rat throughout development using hybridization histochemistry. Messenger RNA is demonstrable in the liver and brown fat from the fetal period onwards and, additionally, in the heart, kidney and gut post-natally. It is proposed that the upregulation of transcription of peroxisomal β oxidation genes in specific tissues follows binding of the receptor to its natural ligand. Thus PPAR may have an important role in cold adaptation and non-shivering thermogenesis as well as in detoxification.

Control of gene expression in tobacco cells using a bacterial operator-repressor system.

We have investigated the efficacy of using the Escherichia coli lac operator‐repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta‐glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator‐free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB‐operator‐gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl‐beta‐D‐thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2‐fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB‐operator‐gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15‐fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor‐‐operator complex and effectively block transcription of a downstream gene.

Characterisation of Kir2.0 proteins in the rat cerebellum and hippocampus by polyclonal antibodies.

Abstract Rabbit polyclonal antibodies were raised to rat Kir2.0 (Kir2.1, Kir2.2 and Kir2.3) inwardly rectifying potassium ion channel proteins. The antibody specificities were confirmed by immunoprecipitation of [35S]-methionine-labelled in vitro translated channel proteins and western blotting. Immunohistochemistry revealed a different patterns of expression of Kir2.0 subfamily proteins in the rat hind-brain (cerebellum and medulla) and fore-brain (hippocampus). Notably, only Kir2.2 protein was detected in the cerebellum and medulla, Kir2.1, Kir2.2 and Kir2.3 proteins were expressed in the hippocampus and immunostaining was not limited to neuronal cell types. Anti-Kir2.1 (fore-brain only) and anti-Kir2.2 (fore- and hind-brain) antibodies showed positive staining in macroglia, endothelia, ependyma and vascular smooth muscle cells. In contrast, anti-Kir2.3 (fore-brain only) immunostaining was limited to neurons, macroglia and vascular smooth muscle. These results indicate that specific regions within the rat fore- and hind-brain have differential distributions of inwardly rectifying potassium ion channel proteins.

Biochemical and Genetic Studies with Regulator Mutants of the Pseudomonas aeruginosa 8602 Amidase System

Surmmary Mutants of Pseudomonas aeruginosa strain 8602 were isolated which were unable to produce an aliphatic amidase (acylamide amidohydrolase, EC 3.5.1.4) and could not grow on acetamide as a carbon or nitrogen source. Amidase-constitutive mutants, producing amidase in the absence of inducing amides, were isolated by selection on succinate+formamide agar. Sixteen mutants were magno-constitutive non-inducible mutants producing amidase at about the same rate or greater than the fully induced wild-type strain. Amidase synthesis in one magno-constitutive mutant was repressed by the non-substrate inducer N-acetylacetamide, but the others were not affected in any way. Six mutants were semi-constitutive, producing amidase at 10–50% of the rate of the magno-constitutive mutants and were induced by N-acetylacetamide. Most of the constitutive mutants were very sensitive to catabolite repression by succinate in pyruvate medium, but succinate produced only partial repression of one magno-constitutive mutant and three semi-constitutive mutants; one semi-constitutive mutant was not repressed except in the presence of inducer. Six mutants isolated from succinate + formamide agar had altered inducer specificity and were induced to form amidase by formamide, which is a very poor inducer for the wild-type strain. The formamide-inducible mutants were also sensitive to catabolite repression by succinate although one mutant was only partially repressed. Phage F 116 was used to transduce the amidase structural and regulator genes. In crosses between constitutive mutants of Pseudomonas aeruginosa as donors and amidase-negative mutants as recipients, the two characters were co-transduced with frequencies of 80–100%. Similarly, in crosses between formamide-inducible and amidase-negative mutants these two characters were co-transduced with frequencies of 89–96%. The amidase structural and regulator genes are considered to be closely linked.

Molecular cloning of two distinct renin genes from the DBA/2 mouse.

We report the molecular cloning of cDNA copies of DBA/2 mouse submaxillary gland (SMG) renin mRNA, which were used to probe Southern transfers of mouse genomic DNA. The results suggested either that there is a single renin gene containing a large intron in that part of the gene corresponding to the probe, or that there are two distinct renin genes. We have shown that the latter is the case by cloning and isolating two similar but distinct renin genes from DBA/2 mouse DNA. Restriction maps of the regions containing the two renin genes are presented, together with nucleotide sequence data locating a complete exon coding for amino acids 268‐315 of mouse SMG renin.

Kidney renin mRNA levels in the early and chronic phases of two-kidney, one clip hypertension in the rat.

The effect of clipping the left renal artery on left and right kidney renin mRNA levels during the early and chronic phases of two-kidney, one clip Goldblatt hypertension in the rat was studied. Renin mRNA levels were determined using northern and dot blotting. Four weeks after clipping, renin mRNA levels were sixfold higher in the left kidney and eightfold lower in the right kidney of the Goldblatt rats compared with the left kidney of the sham-operated rats. Similar analysis at 20 weeks after clipping showed a fourfold increase in the left kidney and a 16-fold suppression in the right kidney compared with age-matched sham-operated control rats. The study demonstrates the profound changes that occur in renin gene expression in the clipped and contralateral kidneys in this model of hypertension and shows that these changes persist into the chronic phase of the hypertension.

Transient transcriptional activation of the IncI1 plasmid anti‐restriction gene (ardA) and SOS inhibition gene (psiB) early in conjugating recipient bacteria

The ardA gene of the enterobacterial plasmid ColIbP‐9 acts to alleviate restriction of DNA by type I systems, while psiB inhibits induction of the bacterial SOS response. Both genes are transferred early in a round of bacterial conjugation as part of the plasmid leading region. We report here that ardA and psiB are transcribed transiently after their conjugative transport into the recipient cell. Transcript levels, monitored by competitive reverse transcription–polymerase chain reaction (RT–PCR) amplification of RNA templates, started to increase about 5 min after the initiation of conjugation in a cell population and probably before the first round of plasmid transfer was completed. Genetic evidence is given that the expression of ardA and psiB is activated when the genes enter the recipient cell on the transferring plasmid strand. It is proposed that these and other leading region genes function to promote the establishment of the immigrant plasmid in the new host and are expressed by transcription from promoters active only in single‐stranded DNA.