Permanent Genetic Resources added to Molecular Ecology Resources Database 1 December 2009-31 January 2010.

This article documents the addition of 220 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Allanblackia floribunda, Amblyraja radiata, Bactrocera cucurbitae, Brachycaudus helichrysi, Calopogonium mucunoides, Dissodactylus primitivus, Elodea canadensis, Ephydatia fluviatilis, Galapaganus howdenae howdenae, Hoplostethus atlanticus, Ischnura elegans, Larimichthys polyactis, Opheodrys vernalis, Pelteobagrus fulvidraco, Phragmidium violaceum, Pistacia vera, and Thunnus thynnus. These loci were cross-tested on the following species: Allanblackia gabonensis, Allanblackia stanerana, Neoceratitis cyanescens, Dacus ciliatus, Dacus demmerezi, Bactrocera zonata, Ceratitis capitata, Ceratitis rosa, Ceratits catoirii, Dacus punctatifrons, Ephydatia mülleri, Spongilla lacustris, Geodia cydonium, Axinella sp., Ischnura graellsii, Ischnura ramburii, Ischnura pumilio, Pistacia integerrima and Pistacia terebinthus.

Cloning and expression analysis of a Petunia hybrida flower specific mitotic-like cyclin.

A cyclin cDNA clone (Pethy;CycB1;1) was isolated from a Petunia hybrida ovary specific cDNA library. Sequence comparison revealed that Pethy;CYCB1;1 protein is highly homologous to mitotic B1 cyclins. Northern analysis and in situ hybridisation experiments showed that its expression is developmentally regulated and restricted to flower organs. We have attempted to define some of the cell division patterns which contribute to shaping each floral organ by analysing Pethy;CycB1;1 expression on Petunia flower sections. While in sepals, epidermis and parenchyma cell division patterns were comparable, there were two distinct cell division patterns in petals. In the epidermis, Pethy;CYCB1;1 expression was found both at the petal tip and along epidermis, whereas in the parenchyma only at the petal tips. In reproductive organs cell divisions were detected only in sporophytic tissues. No signals were detected inside meiotic cells.

Isolation and characterization of a chlorate-resistant mutant of Spirulina platensis.

Three chlorate-resistant mutants of the cyanobacterium Spirulina platensis were obtained by UV irradiation and one of them (LL1) was further characterized for its nutritional requirements and for the capacity to reduce nitrate in vivo and in vitro. The results indicate that mutation leading to chlorate resistance is not due to inactivation of nitrate reductase but is most likely due to the loss of permeability to chlorate and nitrate. The other two mutants seem to have properties similar to those of LL1.

Cloning and characterization of the gene encoding an esterase from Spirulina platensis.

The gene encoding a 23 kDA serine esterase from the cyanobacterium Spirulina platensis has been identified, cloned, characterized and expressed in Escherichia coli. The primary structure of the esterase deduced from the DNA sequence displayed 32% sequence identity with the carboxylesterase (esterase II) encoded by estB of Pseudomonas fluorescens; the highest degree of homology is found in a stretch of 11 identical or highly conserved amino acid residues corresponding to the GXSXG consensus motif found in the catalytic site of many serine proteases, lipases and esterases.

The electrophoretic karyotype of two strains of Candida albicans by transverse alternate field electrophoresis reveals higher number of chromosomes ranging from 1 to 3.5 Mb.

The advent of the powerful electrophoretic technique, pulsed field gel electrophoresis, first developed on the yeast Saccharomyces cerevisiae, has brought a vital impulse to the genetic study on the opportunistic pathogen Candida albicans. We report here on sizing and numbering of Candida chromosomes using transverse alternate field electrophoresis. Our results indicate the occurrence of nine to ten electrophoretic bands (depending on type of Candida strain), that range in approximate size from 1 to 3.5 Mbp, and may account for a higher overall chromosome number, because at least two of these bands appear to be doublets. This number of bands, with smaller size, is considerably higher than previously reported.

Th1 and Th2 cytokine secretion patterns in murine candidiasis: association of Th1 responses with acquired resistance.

Two chemically mutagenized agerminative variants of Candida albicans were used to immunize mice against challenge with highly virulent cells of the parent strain. Although both mutants (Vir- 3 and Vir- 13) resulted in nonlethal infection and could be recovered from mouse organs for many days after the intravenous inoculation of 10(7) to 10(6) cells, significant protection to systemic challenge with virulent C. albicans was induced by only one (Vir- 3) of the two variants. Anticandidal resistance in Vir- 3-infected mice was associated with the occurrence in vivo of strong delayed-type hypersensitivity to Candida antigen, detection in vitro of highly fungicidal effector macrophages, and presence in the serum of a large proportion of Candida-reactive antibodies of the immunoglobulin G2a isotype. Bulk cultures of purified CD4+ lymphocytes from mice infected with either mutant were compared for their ability to produce gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-6 in vitro. After stimulation with specific antigen, CD4+ cells from Vir- 3-immunized mice released large amounts of the Th1-specific cytokines, IFN-gamma and IL-2, at a time when CD4+ cells from Vir- 13-infected mice predominantly secreted the characteristic Th2 cytokines, IL-4 and IL-6. These results were confirmed by quantitative analysis of cytokine-producing Th1 and Th2 cells. In addition, only mice infected with Vir- 3 displayed a high frequency of CD8+ cells with the potential for in vitro lysis of yeast-primed bone marrow macrophages. Purified CD4+ cells from Vir- 3-infected mice, but not a mixture of these cells with CD4+ lymphocytes from mice infected with Vir- 13, could adoptively transfer delayed-type hypersensitivity reactivity onto naive mice. Taken together, these data suggest that both Th1 and Th2 CD4+ lymphocytes may be activated during experimental C. albicans infection in mice.

Mutagenesis of the cyanobacterium Spirulina platensis by UV and nitrosoguanidine treatment.

The production of Spirulina platensis cells resistant to 8-azaguanine or beta-(2-thienyl)-DL-alanine following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and UV-irradiation is described. The conditions for the mutagenesis were determined by monitoring cell viability and the appearance of the two types of mutants as a function of the stage of growth of the tricomes and the length and the conditions of the treatment. The optimal conditions for UV and MNNG mutagenesis were found to be 1-3 min irradiation and 30 min incubation with 50 micrograms MNNG/ml of tricomes derived from cultures entering stationary phase sonicated for 10 s and 5 s respectively. Under these conditions beta-(2-thienyl)-DL-alanine-resistant mutants appeared at a frequency greater than or equal to 10(-4) and greater than or equal to 10(-5) following UV- and MNNG-mutagenesis, respectively. Mutants resistant to 8-azaguanine were found at a frequency approx. 10(-5) only after MNNG mutagenesis. A few chlorate-resistant mutants were also obtained following UV treatment.

Triparental crosses in Streptomyces coelicolor A3(2) as a means of demonstrating the de-repression of SCP1 plasmid conjugative functions.

A conjugative plasmid free in the cytoplasm promotes recombination if it is transferred to two plasmid-less strain in triparental crosses of Streptomyces coelicolor. The recombination frequency is the same as that of biparental crosses between a plasmid-bearing and a plasmid-less strain. The "conjugative" functions of the plasmid are responsible for recombinant formation. Hyphal contact and subsequent heterokaryon formation are necessary but not sufficient steps for the recombinant process. This paper shows that the presence of a conjugative plasmid is necessary for chromosome mobilization and subsequent recombinant formation.

Evidence of heterokaryosis in streptomyces coelicolor A3(2).

Hyphae from mixed cultures of complementary auxotrophs of Streptomyces coelicolor A3(2) did not grow on minimal media (MM) when fertility plasmids ( SCP1 and SCP2) were missing in both strains. The addition of one part per cent of complete medium (CM) to MM allowed growth of vigorous colonies among the tiny colonies of the parental types. The former, amounting to 1%-10% of the total population, turned out to be heterokaryons. They could be propagated on the same medium by plating of hyphal fragments. When five parts per cent of CM were added to MM, beside the heterokaryotic colonies, vigorous 'spindles' of aerial mycelium were formed whenever complementary colonies overlapped. When the SCP1 and SCP2 plasmids were present in one or both parents a clear constraint on the outburst of heterokaryotic aerial mycelium was observed.

Properties of transposon SCTn1 of Streptomyces coelicolor A3(2).

Chloramphenicol resistance (Cmlr) of Streptomyces coelicolor A3(2) behaves like a transposon locus, not being localisable in any region of the map and yet being transferable in crosses at a rate comparable to that of chromosomal markers. It can also be transposed onto a plasmid (SCP1) and back to the chromosome. Some traits, such as arginino-succinate synthase production (ArgG), aerial mycelium formation (AmyA), resistance to tetracycline and to rifamycin C appear to be joined to Cml in three processes: co-mutation, i.e. simultaneous loss, post-mutation, i.e. spontaneous loss at high frequency in subclones from Cmls strains, co-transfer, i.e. joint transfer with the cml locus in crosses or during infection by the aggregate SCP1::SCTn1 plasmid. All these processes have been consistently observed with special attention to the argG locus.

A jumping gene in streptomyces coelicolor A3(2).

The difficulty in mapping the gene for chloramphenicol resistance (cmlR) in Streptomyces coelicolor A3(2) stock strains is possibly due to its location on different sites of the chromosome in various mixed subclones. Fresh isolates from CmlR strains show single unequivocal locations of cmlR. The same holds for CmlR strains derived as revertants from CmlS variants. The two best established sites for cmlR are one between cysA and metA, the other at right of argA, possibly in the right empty arc of the map (Fig. 2). The cmlR gene was assumed to be on a transposon (SCTn1), together with a gene for arginine-succinate synthase (argG), a gene for chromosome transfer (tra) and a gene for aereal mycelium formation (amy). In a CmlR revertant, the cmlR gene appears disjoined from argG (Fig. 5), thus showing the ability of SCTnl to be split and partially transposed. The possible wide occurrence of transposons in the genus Streptomyces is discussed.

Chloramphenicol resistance in Streptomyces coelicolor A3(2): possible involvement of a transposable element.

The transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1- superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1-) it was never displaced by the immigrant chromosome even when the region facing chl+ was selected for. A fraction of the original Chl- mutants presented a requirement for arginine (ArgB-). A Chl- mutant gave rise spontaneously to ArgB- derivatives at high frequency. The same ArgB- requirement come out at high frequency among Chl- derivatives from a cross NFChl- x SCP1-Chl+ in which neither parent required arginine or produced spontaneously arginine-less derivatives. It is suggested that the Chl element is a "transposable element" (Tn) presumably associated with "insertion sequences" (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.

A factor involved in chloramphenicol resistance in Streptomyces coelicolor A3(2): its transfer in the absence of the fertility factor.

An element controlling chloramphenicol resistance (chl) was detected in Streptomyces coelicolor A3(2). Strains sensitive to 1 microgram chloramphenicol ml-1 were obtained among dark scarlet variants. Transfer of the resistance factor was attempted in matings between chloramphenicol-resistant (Chl+) and chloramphenicol-sensitive (Chl-) strains, both of which lacked the SCP1 fertility factor. Transfer of chl was obtained at a much higher rate than that expected for chromosomal markers in SCP1- X SCP1- matings. However, in these particular crosses the latter was also several times higher than usual. All recombinants for chromosomal markers were Chl+. Attempts to locate the chl element failed to distinguish between a chromosomal and an extrachromosomal site. The observed increase in the recombination frequency for chromosomal markers suggests that the chl element may promote recombination.