High-frequency conjugal transfer of a gonococcal penicillinase plasmid.

Gonococci containing a 24 X 10(6)-dalton conjugal plasmid were able to mobilize for transfer a smaller, non-self-transmissible penicillinase (Pcr) plasmid with high frequency under appropriate conditions. In some strains, over 10% of donor colony-forming units transferred the Pcr plasmid in a mating of less than 2 h, which suggests that the conjugal system was naturally derepressed. Colony-opacity variants containing different quantities of an approximately 28,000-dalton outer membrane protein were altered in their ability to act as conjugal donors and recipients. Maximal transfer of the Pcr plasmid was observed between transparent donors and recipients, lacking appreciable amounts of the 28,000-dalton protein. Under conditions of high-frequency Pcr plasmid mobilization, no conjugal mobilization of chromosomal markers could be discerned.     

Members of a Novel Protein Family Containing Microneme Adhesive Repeat Domains Act as Sialic Acid-binding Lectins during Host Cell Invasion by Apicomplexan Parasites

Numerous intracellular pathogens exploit cell surface glycoconjugates for host cell recognition and entry. Unlike bacteria and viruses, Toxoplasma gondii and other parasites of the phylum Apicomplexa actively invade host cells, and this process critically depends on adhesins (microneme proteins) released onto the parasite surface from intracellular organelles called micronemes (MIC). The microneme adhesive repeat (MAR) domain of T. gondii MIC1 (TgMIC1) recognizes sialic acid (Sia), a key determinant on the host cell surface for invasion by this pathogen. By complementation and invasion assays, we demonstrate that TgMIC1 is one important player in Sia-dependent invasion and that another novel Sia-binding lectin, designated TgMIC13, is also involved. Using BLAST searches, we identify a family of MAR-containing proteins in enteroparasitic coccidians, a subclass of apicomplexans, including T. gondii, suggesting that all these parasites exploit sialylated glycoconjugates on host cells as determinants for enteric invasion. Furthermore, this protein family might provide a basis for the broad host cell range observed for coccidians that form tissue cysts during chronic infection. Carbohydrate microarray analyses, corroborated by structural considerations, show that TgMIC13, TgMIC1, and its homologue Neospora caninum MIC1 (NcMIC1) share a preference for α2–3- over α2–6-linked sialyl-N-acetyllactosamine sequences. However, the three lectins also display differences in binding preferences. Intense binding of TgMIC13 to α2–9-linked disialyl sequence reported on embryonal cells and relatively strong binding to 4-O-acetylated-Sia found on gut epithelium and binding of NcMIC1 to 6′sulfo-sialyl Lewis^x might have implications for tissue tropism.     

Male recombination with single and homologous P elements in Drosophila melanogaster

It has previously been shown that, in the presence of a source of P element transposase, male recombination in Drosophila melanogaster is induced at a rate of about 1% in the region of a single P[CaSpeR] element. This paper shows that recombinant chromosomes retain unaltered P[CaSpeR] elements at the original site in a high proportion of cases. This result is incompatible with a simple model in which recombination occurs by resolution of a Holliday junction following P element excision and repair. It has also previously been shown that homozygous regions containing a P element produce male recombination levels of 10–20%, an order of magnitude higher than that given by a single element. This paper shows that reciprocal recombinant chromosomes retaining P[CaSpeR] elements can be combined to produce similarly high levels of recombination. This result potentially allows for recombinant chromosomes from homologous recombination to be analysed at the molecular level in the region of the inserted element.     

Dose dependence of acetylcholinesterase activity in neuroblastoma cells exposed to modulated radio-frequency electromagnetic radiation.

Radio-frequency electromagnetic radiation (RFR) at 915 and 147 MHz, when sinusoidally amplitude modulated (AM) at 16 Hz, has been shown to enhance release of calcium ions from neuroblastoma cells in culture. The dose-response relation is unusual, consisting of two power-density "windows" in which enhanced efflux occurs, separated by power-density regions in which no effect is observed. To explore the physiological importance of these findings, we have examined the impact of RFR exposure on a membrane-bound enzyme, acetylcholinesterase (AChE), which is intimately involved with the acetylcholine (ACh) neurotransmitter system. Neuroblastoma cells (NG108), exposed for 30 min to 147-MHz radiation, AM at 16 Hz, demonstrated enhanced AChE activity, as assayed by a procedure using 14C-labeled ACh. Enhanced activity was observed within a time window between 7.0 and 7.5 h after the cells were plated and only when the exposure occurred at power densities identified in a previous report as being effective for altering the release of calcium ions. Thus RFR affects both calcium-ion release and AChE activity in nervous system-derived cells in culture in a common dose-dependent manner.     

T cell receptor alpha-chain influences reactivity to Mls-1 in V beta 8.1 transgenic mice.

Most, but not all, V beta 8.1+ T cells respond to M1s-1 and are clonally deleted in the thymus of M1s-1-expressing animals. To formally examine the role of the TCR alpha-chain in reactivity and tolerance to M1s-1, we have analyzed M1s-1 reactivity in a large panel of CD4+ hybridomas generated from TCR V beta 8.1 transgenic mice, that express an identical, potentially M1s-1-reactive beta-chain. The data show that the alpha-chain strongly influences the M1s-1 reactivity of the hybridomas and that the differences in reactivity had relevance for tolerance. Thus, V alpha 11+ hybridiomas were biased toward M1s-1 reactivity and V alpha 11+ T cells were correspondingly absent from the peripheral repertoire of M1s-1-expressing transgenic mice. V alpha 2+ hybridomas, on the other hand, were biased against M1s-1 reactivity, and V alpha 2+ T cells were correspondingly amplified in the M1s-1-expressing transgenic mice. Structural analysis of the alpha-chains revealed that the M1s-1 reactivity of the V alpha 11+ hybridomas segregated precisely with family member, such that V alpha 11.1+ hybridomas were M1s-1-reactive and V alpha 11.3+ hybridomas were not M1s-1-reactive. On the other hand, there was not a clear correlation between family member and M1s-1 reactivity in the V alpha 2+ hybridomas. The hybridomas also showed striking variation in their reactivity to staphylococcal enterotoxin B (SEB), and the SEB reactivity of the V alpha 11+ hybridomas correlated precisely with family member and with M1s-1 reactivity. In contrast, there was not a clear correlation with V alpha 2+ alpha-chain structure and SEB reactivity. Also, there was no correlation between M1s-1 reactivity and SEB reactivity in individual V alpha 2+ hybridomas, suggesting that the recognition of the two superantigens by the same TCR is not equivalent. Taken together, these data define a role for the TCR alpha-chain in superantigen reactivity and T cell tolerance, and provide a structural explanation for the different fates of M1s-1-reactive T cells in normal and transgenic mice.     

Mutation to erythromycin dependence in Escherichia coli K-12

A nitrosoguanidine-induced mutant of Escherichia coli K-12 strain JC12 was absolutely dependent on erythromycin or related macrolide antibiotics for growth. The only other drugs which permitted growth (lincomycin and chloramphenicol) are, like the macrolides, inhibitors of the 50S ribosome. The order of relative effectiveness of these drugs was macrolides > lincomycin > chloramphenicol. Rates of growth with all drugs were concentration dependent. Erythromycin starvation was followed by normal rates of increase in cell mass and macromolecular synthesis for approximately one mass-doubling time, after which macromolecular synthesis abruptly ceased and cell lysis and death occurred. The dependent mutant gave rise spontaneously to revertants to independence with very high frequency (10(-4)). The gene (mac) for macrolide dependence is located near minute 25 on the E. coli chromosome; it does not result in increased resistance to these drugs. A separate gene for erythromycin resistance (eryA) is located in the cluster of ribosomal structural genes near spc, close to minute 63. Dependence on macrolides was most clearly evident in strains carrying mutations at both eryA and mac.     

The genetic architecture of parallel armor plate reduction in threespine sticklebacks

How many genetic changes control the evolution of new traits in natural populations? Are the same genetic changes seen in cases of parallel evolution? Despite long-standing interest in these questions, they have been difficult to address, particularly in vertebrates. We have analyzed the genetic basis of natural variation in three different aspects of the skeletal armor of threespine sticklebacks (Gasterosteus aculeatus): the pattern, number, and size of the bony lateral plates. A few chromosomal regions can account for variation in all three aspects of the lateral plates, with one major locus contributing to most of the variation in lateral plate pattern and number. Genetic mapping and allelic complementation experiments show that the same major locus is responsible for the parallel evolution of armor plate reduction in two widely separated populations. These results suggest that a small number of genetic changes can produce major skeletal alterations in natural populations and that the same major locus is used repeatedly when similar traits evolve in different locations.