Use of a modified Escherichia coli trpR gene to obtain tight regulation of high-copy-number expression vectors.

It has been found that with high-copy-number vectors utilising the Escherichia coli trp promoter the amount of repressor protein produced from the single chromosomally located trpR gene is inadequate for tight repression to be obtained. An attempt has been made to overcome this problem by inserting the trpR gene in cis into the expression vector. This proved unsuccessful because transcription from the trp promoter of such a plasmid could not be induced with 3,beta-indole acrylic acid, probably because the trpR gene is autogenously regulated. However, it was found that when the natural trpR promoter was replaced with a relatively weak constitutive promoter a useful self-repressible vector could be formed. A modified trpR gene of this type has been used to obtain tightly controlled expression of human interferon-beta (IFN-beta) from a vector having a copy number of 400. Tight regulation is particularly important in this case as IFN-beta is highly toxic to the E. coli cell.

Iliac crest bone biopsy for diagnosis of aluminum toxicity and a guide to the use of deferoxamine.

Diagnosis of aluminum-related bone disease in patients with renal failure often requires a bone biopsy. Percutaneous biopsy of the iliac crest has been safely carried out as an outpatient procedure in over 300 patients. No instances of significant bleeding or infection have occurred. The procedure was well accepted in 85% to 90% of patients recently surveyed. Before biopsy, each patient received two short courses of oral tetracycline, separated by a ten- to 14-day interval. Double tetracycline labeling permits the evaluation of dynamic characteristics of bone for the diagnosis of aplastic disease due to aluminum toxicity. When a diagnosis of aluminum toxicity is established, treatment with the chelating agent deferoxamine (DFO) administered during dialysis is effective for aluminum removal. Initially, a standardized infusion of DFO, 40 mg/kg, is administered over two hours immediately following dialysis treatment. The increase in plasma aluminum 24 to 48 hours following such an infusion provides an index of tissue aluminum stores and provides a guide to the appropriate therapeutic dose of DFO. DFO (2 to 4 g) is administered once weekly during the last two hours of dialysis; in rare cases higher dosages have been used. Reported side effects of DFO therapy include hypotension, hypoferremia, and allergic reactions; other investigators have reported ocular abnormalities and unusual infections. Patients are reevaluated after 6 to 12 months of therapy with a DFO infusion test and/or bone biopsy. However, the duration of therapy needed for the successful treatment of aluminum-related bone disease is uncertain.

Pseudohypopyon in Best's vitelliform macular dystrophy.

We examined ten eyes with pseudohypopyon in seven patients with Best's vitelliform macular dystrophy. Fluorescein angiography showed hyperfluorescent defects in the retinal pigment of the superior half of the lesion in all ten eyes. The electro-oculographic findings were abnormal in all four patients who underwent this test. The fluid shifted slightly in two of six patients whose heads were turned to the side for an hour or longer, indicating that the material was probably located between the retinal pigment epithelium and the sensory retina. The volume of the hypopyon increased in one eye.

A conjugative 'plasmid' lacking autonomous replication.

Attempts were made to isolate open and covalently closed circular DNA from strains containing the IncJ plasmids. All of the methods tried were unsuccessful. It was shown that the IncJ plasmid R391 can integrate into the Escherichia coli K12 chromosome and can mobilize chromosomal markers from a single origin in an orientated manner. It is proposed that the IncJ plasmids are integrated in the chromosome for most, if not all, of their existence and this explains the inability to isolate plasmid DNA from strains containing them.

Transposable gentamicin resistance in IncW plasmids from Hammersmith Hospital.

A transposon, Tn733, encoding the gentamicin acetyltransferase AAC(3) was found on two gentamicin R plasmids of IncW at Hammersmith Hospital. Transposon TN733 has a molecular mass of 5.8 megadaltons and gives a characteristic 2.4 megadalton fragment on digestion with EcoRI. The appearance of gentamicin resistance on a transposon will increase the chances of spread of this gene.

Recombinant plasmids formed in vivo carrying and expressing two incompatibility regions.

The formation in vivo of recombinants between a plasmid of incompatibility group N (R1010-10) and plasmids of groups P (R751) and W (R388) is described. From examination of the molecular weights of these recombinant plasmids, they appear to be cointegrates. These cointegrates have the incompatibility properties of both 'parent' plasmids.

The nature of the genetic determinant for the SHV-1 beta-lactamase.

In two unrelated plasmids of incompatibility groups FII and N the gene for the SHV-1 beta-lactamase exists as part of a transposable element of molecular weight 9.5 megadaltons. This transposon has moved onto plasmids of at least three incompatibility groups; PI, Ialpha and J. This confirms the suggestion that the recent spread of the SHV-1 beta-lactamase has been associated with the transposition of the genetic determinant of this enzyme between unrelated plasmids.

Characterization and incompatibility properties of ROM- derivatives of pBR322-based plasmids.

We have characterized a copy number mutant of the pBR322-based plasmid pWT111. A single nucleotide transversion in loop II' of RNAI results in an eightfold increase in plasmid copy number. Removal of the rom coding region from pWT111 cop results in a further sixfold increase in copy number. We present evidence that ROM is involved in the strong incompatibility effect seen between pMB1 and ColE1 type plasmids.

Naturally occurring plasmids in Acinetobacter calcoaceticus: pAV2, a plasmid which influences the fertility of the sex factor pAV1.

Acinetobacter calcoaceticus strain EBF65/65 harbours a cryptic plasmid, pAV2, which has been shown by electrophoretic separation on agarose gels to have a molecular mass of approximately 13.5 megadaltons (Md). Transfer of the previously described sex factor pAV1 (Hinchliffe & Vivian, 1980 a,b) from the hospital strainJC17 into strains possessing pAV2 occurs only at a low frequency, whereas transfer to similar strains lacking pAV2 occurs at a much higher frequency. In EBF65/65, pAV1 may be present in strains possessing or lacking pAV2; pAV1 strains lacking pAV2 correspond to strains previously described as pAV1a (Hinchliffe & Vivian, 1980b) whereas pAV1 strains which also possess pAV2 correspond to pAB1b strains. The genetic evidence presented here is consistent with the hypothesis that pAV2 specifies a host restriction and modification system that is active against pAV1. Physical evidence from agarose gel electrophoresis indicates that pAV1 corresponds to a band of approximately 85 Md in strain JC17. The corresponding band in strains of EBF65/65 is difficult to distinguish because of the presence of a further cryptic plasmid band of approximately 88 Md, designated pAV3. A small cryptic plasmid of approximately 6 Md, designated pAV4, is reported for EBF65/65.

A transposon, Tn732, encoding gentamicin/tobramycin resistance.

Gentamicin and tobramycin are important antibiotics in the treatment of hospital infections because of their activity against a wide range of bacterial genera. With their increasing use, bacteria resistant to these drugs have appeared, the resistance being frequently plasmid determined. The resistance genes determine various enzymes that modify and inactivate the drugs and there is association between particular gentamicin/tobramycin resistance genes and plasmids of particular groups, implying that acquisition of such a gene by any plasmid is a rare event. We now report the identification of a transposon or 'jumping gene' encoding the gentamicin/tobramycin adenylylating enzyme, ANT(2"), on a plasmid of incompatiblity group FII (IncFII).

pHH502, a plasmid with IncP and IncI alpha characters, loses the latter by a specific recA-independent deletion event.

Plasmid pHH502, of molecular weight 70 X 10(6), determined resistance to tetracycline, chloramphenicol, trimethoprim, sulphonamides and mercuric chloride and was incompatible with members of IncP and IncI alpha. It resembled other plasmids of IncI alpha in the following properties: it determined pili that were morphologically and serologically I alpha pili, whose production was repressed in established plasmid-carrying (R+) cultures; its transfer was equally efficient in liquid or on solid medium; it exerted surface exclusion against other IncI alpha plasmids; it was non-transferable to Proteus. In a reproducible, recA-independent event, pHH502 gave rise to pHH502-1, a plasmid of molecular weight 40 X 10(6), lacking determinants for resistance to tetracycline and chloramphenicol and all detectable IncI alpha characteristics. pHH502-1 was incompatible only with IncP plasmids and resembled other IncP plasmids in determining constitutive production of rigid pili, in its surface exclusion, in transferring at greater frequency on solid than in liquid medium and in being transmissible to Proteus mirabilis. It differed from other IncP plasmids in the morphology and serological type of its pili and in failing to transfer to Pseudomonas aeruginosa or Acinetobacter calcoaceticus. Small numbers of pHH502-1 rigid pili were present on bacteria carrying pHH502. Possible mechanisms for the generation of pHH502 and pHH502-1 are discussed.