Transposition of DNA inserted into deletions of the Tn5 kanamycin resistance element

Summary Tn5-trp hybrid transposons have been constructed by insertion of a trpPOED Hind III fragment into an in vivo Tn5 internal deletion mutant or by substitution of trp for the internal Tn5 Hind III fragment. These hybrids are called, respectively, Tn409 and Tn410. Both Tn409 and Tn410 will transpose into in the presence of a complementing Tn5 element. In the absence of a wild Tn5, lysogens carrying R1162::Tn409 and R1162::Tn410 plasmids will yield trp phages at less than six per cent of the complemented frequency. This reduction indicates that Tn409 and Tn410 lack a diffusible transposition function provided by wild Tn5 elements. However, the formation of trp phages without complementation is real. Most of these transducing particles contain Tn409 and Tn410 still linked to the carrier R1162 plasmid. This observation suggests that uncomplemented Tn409 and Tn410 elements mediate the formation of -transposon-plasmid cointegrate structures. Thus, the missing transposition function may be involved in resolving these cointegrate structures to the final ::Tn409 or ::Tn410 product.Abbreviations p.f.u. plaque-forming units - MIC minimal inhibitory concentration - LFT low frequency transducing - HFT high frequency transducing     

Size, maturation and the social control of sex reversal in the coral reef fish Anthais squamipinnis

A major hypothesis to explain the causal initiation of protogynous sex reversal is that females change sex upon reaching a critical size. A study of the coral reef fish Anthias squamipinnis shows that the size hypothesis does not hold. Females from two neighbouring, but spatially discrete and probably genetically homogeneous populations on Aldabra Island changed sex at distinctly different sizes. Previous laboratory and field studies in which sex reversal has followed the removal of a male from social groups have been uncontrolled and thus permit the interpretation that sex reversal is caused by non-specific social disruption or by causes other than male removal. In this study, a male was removed from each of eleven single-male and five multi-male social groups in the laboratory ( N = 8 male removals) and in the field ( N = 19 male removals). In each group, the result was that one female changed sex. Laboratory controls made it unlikely that sex reversal was induced by non-specific disruption and field observations showed that sex reversals resulted from male removals and were not coincidental, ongoing events. Previous statements that sex change is controlled by the presence or absence of a male, by inhibition of a female's tendency to change sex, or by aggression or dominance are shown, by an analysis of the complexity of issues, to be premature. Gonadal histology on 130 specimens confirmed that this species is a monandric, protogynous hermaphrodite and provided details of gonadal transformation.     

Generation of lipopolysaccharide-induced interferon in spleen cell cultures

Incubation of murine spleen-cell cultures with lipopolysaccharide (LPS) induces interferon (IF) production. Maximal IF levels are obtained after incubation with 100 g/ml for 10 h. Two inbred mouse strains differing in their ability to generate LPS-induced IF in spleen-cell cultures were used: C3H/eB, which generates high levels of IF (about 60 units/ml), and C3H/HeJ, which fails to generate detectable quantities of IF. In a genetic analysis these strains were hybridized and IF production was determined in spleen-cell cultures from F₁ and F₂ generations, and from backcrosses of F₁ hybrids to parent strains. The results indicate that, in parent strains, a single dominant autosomal gene is responsible for differences in IF production in spleen cultures. LPS-induced IF in spleen-cell cultures resists pH 2 for as long as 48 h, but is labile to heating at 56° C for 30 min. Both macrophages and lymphocytes must be present in cultures for generation of LPS-induced IF. By using mixed cultures of macrophages and lymphocytes from C3H/eB and C3H/HeJ mice, it was shown that macrophages have to interact directly with LPS to enable IF production in the cultures.     

The role of serine hydroxymethyltransferase in cell proliferation: DNA synthesis from serine following mitogenic stimulation of lymphocytes

It is shown that the time-course of incorporation of radioactivity from [3-¹⁴C]serine into nucleic acids parallels DNA synthesis following mitogenic stimulation of human peripheral blood lymphocytes by phytohaemagglutinin (PHA). The activity of serine hydroxymethyltransferase was elevated about four-fold in PHA-stimulated lymphocytes compared to that in unstimulated control ceils. It is suggested that lymphocytes, in common with other proliferating cell systems:, may synthesize serine de novo for utilization in pathways of nucleotide biosynthesis following mitogenic stim--ulation.     

Flow cytometric estimation of DNA and RNA content in intact cells stained with Hoechst 33342 and pyronin Y

The addition of RNA content estimation to flow cytometric measurement of DNA content provides valuable information concerning cells' transitions between quiescent and proliferative states. Equilibrium staining methods employing acridine orange have been used for DNA/RNA content measurement but are difficult to apply to intact cells and impractical for use in conjunction with fluorescent antibodies or ligands for demonstration of cell surface structures. I have used a combination of Hoechst 33342 (HO342) and pyronin Y (PY) to stain intact cells for DNA/RNA content estimation with a dual source flow cytometer using UV and blue-green or green excitation, measuring HO342 fluorescence at 430--470 nm and PY fluorescence at 590--650 nm. Results obtained with cultured cells and stimulated lymphocytes are in good agreement with those obtained using acridine orange for DNA/RNA staining; about half of the PY fluorescence can be removed from ethanol-fixed cells stained with HO342 and PY by RNAse digestion. The HO342/PY method can be combined with fluorescein immunofluorescence for detection of cell surface markers. HO342 can be combined with other tricyclic heteroaromatic dyes for DNA/RNA estimation; the combination of HO342 and oxazine 1 can be excited in a dual source instrument using a mercury arc lamp and a helium-neon laser. The staining procedure is simple; cells in medium are incubated with 5 microM HO342 at 37 degrees C for 45 min, 5 microM PY (or oxazine 1) is then added and cells are analyzed without washing after an additional 45 min incubation. Suitability of these dye combinations for vital cell staining and sorting remains to be determined.     

Predation and space utilization patterns in a marine epifaunal community

The relative abundance patterns of several sessile epifaunal species occurring subtidally on large artificial substrata (pilings) were examined under experimental conditions involving the manipulation of densities of the echinoid Arbacia punctulata (Lamarck). The foraging activities of this predator could denude the substratum of most species with notable exceptions including the colonial hydroid Hydractinia echinata Fleming and the sponge Xestospongia halichondroides (Wilson). Moreover, these were the only two of the twenty most common species which did not significantly change in relative abundance over the experimental period. Both species had low recruitment rates and were commonly associated with substrata which had been submerged for several years. Neither species aggressively interacted with adjacent spatial competitors but instead, appeared to employ a defensive space utilization ‘strategy’. Provision of unoccupied substrata by Arbacia was apparently the major factor favoring both recruitment and growth of Hydractinia, which covered up to 30% of the area on the oldest pilings. More recently submerged substrata were covered by species such as Schizoporella errata (Waters) which had a much higher recruitment rate but was commonly overgrown by several other species. Recruitment rate, competitive ability, and vegetative growth are discussed in terms of the size of the substratum and the possibility of biased sampling in fouling studies. The widespread introduction of large artificial substrata into the natural environment has considerably altered the structure of the natural habitat and constitutes a potentially important selective force for changes in settlement preferences, especially among species such as Hydractinia which persist and become abundant on these substrata.     

Separation of urine proteins on the anion-exchange resin mono QTM

Proteins excreted in urine due to renal failure were separated on Mono Q^TM, a new strong anion exchanger designed for fast high-resolution protein separations. The separation procedure was divided into two steps. The first step involved removal of low-molecular- weight substances by rapid desalting on a Sephadex G-25 Superfine column. In the second step, the total protein fraction (3–6 ml) was loaded onto the Mono Q column with the aid of a superloop. The proteins were adsorbed onto the top of the ion-exchanger column and gradually displaced by a combined pH and salt gradient in 40 min. The choice of ion exchanger and initial operating conditions were based on data obtained from electrophoretic titration curve experiments. Identification of separated proteins was achieved by fused rocket electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, respectively.     

C-n.m.r. spectral study of C reductively methylated glycopeptides derived from glycophorin A

¹³C-n.m.r. spectral data for ¹³C reductively methylated intact homozygous and heterozygous glycophorins A were compared with the ¹³C-n.m.r. spectral data for the ¹³C reductively methylated homozygous and heterozygous N-terminal glycopeptides derived from the trypsin digest of glycophorin A. The results indicate that pronounced aggregation of this glycoprotein in solution does not affect the structural differences that we have previously observed for glycophorins A^M and A^N at and/or near the N-terminal amino acid. Moreover, the data suggest that two structural states exist for glycophorin A^M.     

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.     

Human 92- and 72-kilodalton type IV collagenases are elastases.

Elastin is critical to the structural integrity of a variety of connective tissues. Only a select group of enzymes has thus far been identified capable of cleaving insoluble elastin. Recently, we observed that human alveolar macrophages secrete elastase activity that is largely inhibited by the tissue inhibitor of metalloproteinases (TIMP). This finding suggested that one or more of the metalloproteinases released by alveolar macrophages has elastase activity. Accordingly, we tested pure human interstitial collagenase, stromelysin, 92-kDa type IV collagenase, and 72-kDa type IV collagenase for elastolytic activity using kappa-elastin zymography and insoluble 3H-labeled elastin. The 92- and 72-kDa type IV collagenases were found to be elastolytic in both assay systems. A recombinant preparation of 92-kDa type IV collagenase with gelatinolytic activity was also found to be elastolytic. Organomercurial activation was essential to detect elastolytic activity of the native 92- and 72-kDa type IV collagenases and enhanced the elastase activity of the recombinant 92-kDa enzyme. On a molar basis the recombinant 92-kDa type IV collagenase was approximately 30% as active as human leukocyte elastase in solubilizing 3H-labeled elastin. Exogenously added TIMP in significant molar excess abolished the elastase activity of the 92- and 72-kDa type IV collagenases. Stromelysin and interstitial collagenase showed no significant elastolytic activity, although both were catalytically active against susceptible substrates. Conditioned media from cultures of human mononuclear phagocytes containing the 92-kDa enzyme produced a distinct zone of lysis in the kappa-elastin zymograms at this molecular mass. These results definitively extend the spectrum of human proteinases with elastolytic activity to metalloproteinases and suggest the enzymatic basis for elastase activity observed with certain cell types such as human alveolar macrophages.