Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions.

We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 µg of vector DNA and 10(6) protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

The psychosocial experience of women treated for breast cancer by high-dose chemotherapy supported by autologous stem cell transplant: a qualitative analysis of support groups.

Autologous bone marrow transplantation (AuBMT) is probably among the most aggressive of physical treatments endured by cancer patients. High-risk breast cancer patients who choose this therapy face prolonged, agonizing and life-threatening interventions that are no less arduous than confronting the malignant disease itself. The study, which aimed to broadening our understanding of the psychosocial impact and the implications of AuBMT, presents a protocol analysis of group support intervention in 45 recipients (eight to ten women in five groups). The sessions were held at the Transplant Department at the Chaim Sheba Medical Center. The contribution of group support to the healing process was examined. The findings show that recovery was affected by a wide range of psychosocial factors, specifically highlighting the impact of transplantation and survival on five domains, viz. physical, psychological/emotional, vocational, social and family/spousal intimacy. Illness and treatment management is also discussed. The support generated by the group, both individually and collectively, was found to contribute significantly to the spectrum of resources available to the participants.

Alternating patient position following the induction of obstetric epidural analgesia does not affect local anaesthetic spread.

To assess the influence of gravity on epidural-induced sensory blockade during the first stage of labour, 60 parturients were recruited to a randomized, controlled, single-blinded study. In all cases an epidural catheter was placed with the patient in the left lateral position. Thereafter, patients were randomized to one of three groups. In group supine (S), the epidural injection was performed in the supine position with a 15-20 degrees right flank elevation and the patient remained supine. In group left (L) patients remained in the left lateral position for 10 min after injection. In group left and right (L+R) patients remained in the left lateral position for 5 min and then assumed the right lateral position for 5 min. Thereafter, in groups L and L+R patients lay supine with a 15-20 degrees right flank elevation. All patients received 50 microg fentanyl in 10 ml of 0.125% bupivacaine. Ten minutes after injection loss of pinprick and temperature sensation were assessed and pain scores measured. Height of sensory deficit 10 min after injection and after maximal blockade were unaffected by patient position. In all cases maximal blockade reached T8 or higher. Time to maximal block was 14.7 +/- 2.3 min, 14.1 +/- 1.6 min and 13.9 +/- 1.5 min for groups L, L+R and S respectively. After maximal blockade, pain scores were significantly (P<0.0001) decreased in all groups, with no differences between them. We conclude that position immediately following local anaesthetic drug administration has little effect on onset and distribution of epidural analgesia.

Environmental conditions differentially affect vir gene induction in different Agrobacterium strains. Role of the VirA sensor protein.

The induction of vir gene expression in different types of Agrobacterium strains shows different pH sensitivity profiles. The pH sensitivity pattern demonstrated by octopine Ti strains was similar to that of a supervirulent leucinopine Ti strain, whereas this was different from that shown by nopaline Ti strains and agropine Ri strains. Data are given which indicate that these differences are due to different properties of the virA genes of these wild types. An exceptional case was formed by strains with the limited-host-range plasmid pTiAG57 which showed AS-dependent vir induction only if reduced inoculum sizes were used and the temperature was 28 degrees C or below.

The Agrobacterium tumefaciens T-DNA gene 6(b) is an onc gene.

In this article it is shown that the T-DNA of Agrobacterium tumefaciens contains besides the well-known cyt and aux genes another gene with an oncogenic effect in plants. The gene in question is called 6(b) and causes the formation of small tumors in plant species such as Nicotiana glauca and Kalanchoe tubiflora.

Chromosomal nodulation genes: Sym-plasmid containing Agrobacterium strains need chromosomal virulence genes (chvA and chvB) for nodulation.

The chromosomal genes chvA and chvB of Agrobacterium tumefaciens, which mediate attachment to plant cells, were found to be essential not only for tumour induction but also for the formation of root nodules on plants.

Expression of a Rhizobium phaseoli Sym plasmid in R. trifolii and Agrobacterium tumefaciens: incompatibility with a R. trifolii Sym plasmid.

Identification of the Sym plasmid in Rhizobium phaseoli strain RCC3622 is described. Introduction of this plasmid into R. trifolii or Agrobacterium tumefaciens strains resulted in bacteria capable of forming characteristic spherical root nodules on beans. This Sym plasmid, designated pSym9, was characterized as 275 MDa and nonconjugative. pSym9 was incompatible with the R. trifolii Sym plasmid pSym5, and carries genes determining a melanin-like black pigment. A second plasmid of 135 MDa, pRph3622a, was also transferred from R. phaseoli to R. trifolii and A. tumefaciens. Transconjugants carrying this plasmid did not form root nodules on beans. In contrast to other Rhizobium plasmids, pRph3622a was unstable in A. tumefaciens.

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants.

Transposon-insertion mutants with vir- Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir- nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir- octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir- octopine Ti plasmid mutations and vir- nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO- mutants produced rooty tumors on Kalanchoë tubiflora.

Phenotypic expression of mutations in a wide-host-range R plasmid in Escherichia coli and Rhizobium meliloti.

Eight different derivatives of R plasmid RP1 with thermosensitive mutations affecting maintenance in Escherichia coli and Pseudomonas aeruginosa were introduce into Rhizobium meliloti. None of the plasmids showed a thermosensitive character in R. meliloti. On the other hand, a certain deletion mutation in RP1 was found to cause plasmid instability in rhizobia and agrobacteria, but not in E. coli.

Tumor formation and rhizogenicity of Agrobacterium rhizogenes carrying Ti plasmids.

Octopine Ti plasmids were introduced into different Agrobacterium rhizogenes strains. Transconjugants became able to utilize octopine and to induce aberrant tumors, the surface of which was all covered wioth roots of different size and morphology. The plasmids endogenous in A. rhizogenes were shown to be compatible with incRH-1 Ti plasmids.

Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens.

Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become established only infrequently in recipients with a nopaline Ti plasmid and, vice versa, nopaline Ti plasmids were only rarely established in recipients with an octopine Ti plasmid. Rare clones in which the incoming octopine (nopaline) Ti plasmid had been established despite the presence of a nopaline (octopine) Ti plasmid appeared to harbor cointegrates consisting of the entire incoming Ti plasmid and the entire resident Ti plasmid. The integration event invariably had occurred in a region of the plasmids that is highly conserved in evolution and that is essential for oncogenicity. These results show that octopine and nopaline Ti plasmids cannot be maintained as separate replicons by one and the same cell. Therefore, be definition, these plasmids belong to the same incompatibility group, which has been names inc Rh-1. Agrobacterial non-Ti octopine and nopaline plasmids were found to belong to another incompatibility group. The tumorigenic properties of strains harboring two different Ti plasmids, in a cointegrate structure, were indicative of the virulence genes of both of them being expressed. The agrobacterial non-Ti octopine and nopaline plasmids did not influence the virulence properties encoded by the Ti plasmid.