Compartmentalization of the proteins encoded by IS50R

IS50R is a transposable genetic element that serves as the right inverted repeat of the transposon Tn5. Earlier work has shown that IS50R encodes at least two proteins (called P1 and P2) involved in transposition. In this paper, we describe the localization properties of the proteins encoded on this repeat. Strains were constructed that overproduced either these two proteins or hybrids between beta-galactosidase and the IS50R proteins. An antiserum was raised against the hybrid proteins, and this was used to study the localization of P1 and P2. Based on studies in maxicells as well as in growing cells, we show that P1 and P2 are localized differently in the cell. P2 is a cytoplasmic protein, while P1 largely fractionates with the membrane.     

Role of the IS50 R proteins in the promotion and control of Tn5 transposition

IS50R, the inverted repeat sequence of Tn5 which is responsible for supplying functions that promote and control Tn5 transposition, encodes two polypeptides that differ at their N terminus. Frameshift, in-frame deletion, nonsense, and missense mutations within the N terminus of protein 1 (which is not present in protein 2) were isolated and characterized. The properties of these mutations demonstrate that protein 1 is absolutely required for Tn5 transposition. None of these mutations affected the inhibitory activity of IS50, confirming that protein 2 is sufficient to mediate inhibition of Tn5 transposition. The effects on transposition of increasing the amount of protein 2 (the inhibitor) relative to protein 1 (the transposase) were also analyzed. Relatively large amounts of protein 2 were required to see a significant decrease in the transposition frequency of an element. In addition, varying the co-ordinate synthesis of the IS50 R proteins over a 30-fold range had little effect on the transposition frequency. These studies suggest that neither the wild-type synthesis rate of protein 2 relative to protein 1 nor the amount of synthesis of both IS50 R proteins is the only factor responsible for controlling the transposition frequency of a wild-type Tn5 element in Escherichia coli.     

Orientation of IS50 transposase gene and IS50 transposition.

Reversal of transposase gene orientation with respect to the nonidentical ends of IS50 strongly decreased IS50 transposition in both Dam- and Dam+ hosts. In either orientation, IS50 transposase expression was unaffected. These effects were independent of the surrounding DNA context. This shows that the efficiency of IS50 transposition is dependent on transposase gene orientation. The transposition frequencies of transposons utilizing inverted IS50 inside ends (IE), IE-IE transposons, were lower than either outside end (OE)-IE or OE-OE transposons.     

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1–P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix–turn–helix domain (amino acids I13–R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1–D12) did not bind. The N-terminal M1–P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1–M234) and Tnp26 M1–P56 fragment. These findings indicate that the helix–turn–helix and disordered domains of Tnp26 play a role in Tnp26–TIR complex formation. Both domains are conserved in all members of the IS26 family.     

Membrane association with multiple calcium ions: vitamin-K-dependent proteins, annexins and pentraxins.

Peripheral membrane association with high calcium stoichiometry is shared by three families of proteins: annexins, pentraxins and vitamin-K-dependent proteins. Recent crystal structure determinations, biophysical studies of membrane binding and analyses of protein electrostatic properties offer striking and different concepts for membrane association by each of these protein families.     

recA-independent recombination between repeated IS50 elements is not caused by an IS50-encoded function.

Certain pBR322-related plasmids containing direct repeats of the insertion element IS50 appear to be unstable in recA Escherichia coli because smaller recombinant derivatives accumulate rapidly in plasmid DNA populations. We show here that (i) this instability is plasmid specific, but not IS50 specific; (ii) it is due to a detrimental effect exerted by these plasmids on bacterial growth; and (iii) the growth impairment is alleviated in cells harboring the smaller recombinant plasmids. Although a recent report had concluded that accumulation of recombinants reflected an IS50-specific recombination function, when correction is made for the relative growth rates of cells containing the parental and recombinant plasmids the evidence for such a recombination function disappears.     

IS50-mediated inverse transposition. Discrimination between the two ends of an IS element

The crystal and molecular structure of l-pyroglutamyl-β-(2-thienyl)-l-alanyl-l-prolinamide, < Glu-Thi-Pro-NH₂(Thi²-TRH), C₁₇H₂₂N₄O₄S, has been determined from X-ray diffraction data. Thi²-TRH is a highly active analogue of thyroliberin, a thyrotropin-releasing hormone (TRH), in which the imidazole ring of the central histidine moiety in the natural hormone has been replaced by a 2-thienyl group. Thi²-TRH crystallizes from water in the monoclinic space group P2₁, $\text{a = 9.340(1)}\text{A}\text{?}\text{, b = 21.961(3)}\text{A}\text{?}\text{, c = 9.449(1)}\text{A}\text{?}$ and β = 109.58(1) °, with two molecules per asymmetric unit. These independent molecules, A and B, have the same general backbone conformation with the φ₂, ψ₂ and ψ₃ torsional angles close to ?90 °, +120 ° and +150 °, respectively, but they show different magnitudes of rotational disorder in the thiophene ring as well as a certain disorder in the pyrrolidine ring. A and B are cross-linked by four interchain hydrogen bonds, forming a two-stranded antiparallel β-pleated sheet structure. The molecules in these dimer fragments are further hydrogen-bonded to successive translated molecules along the a and c axes, forming a pronounced two-dimensional predominantly hydrophobic layer structure. These layers, in which the atoms are almost equally arranged on both sides, are separated by ordinary van der Waals' distances. A close correlation between the molecular conformation in the solid state and the preferential conformation in solution is found. It is concluded that the crystalline structure of Thi²-TRH possesses structural features which may be of relevance in the hormone-receptor interaction process.     

Membrane proteins on the move.

The Keystone Symposium on Membrane protein structure/function relationships was held on 5-11 March 2001 in Tahoe City, California, USA.     

Transposition of IS50L activates downstream genes.

A transposition system constructed to detect the transposition of Tn5 to a site upstream of the lacZ gene has revealed that transposition of IS50L can activate downstream genes. Expression is apparently mediated by the NPTII promoter. Transposase produced either by IS50R or by the suppressed IS50L catalyzed transposition of IS50L.     

Membrane proteins in senescent erythrocytes.

The examination of erythrocyte senescence has been facilitated by recent advances in techniques for the isolation of aged red cells. One of these methods, which uses biotinylated rabbit erythrocytes, has been used to examine the state of membrane proteins in effete cells. These aged red cells were found to have normal ratios of alpha-spectrin and beta-spectrin as well as normal levels of ankyrin. The observation concerning ankyrin is particularly important due to the sensitivity of this protein to proteolysis and the postulated action of proteinases in the aging process. The senescent erythrocytes were also found to have an altered ratio of bands 4.1a and 4.1b without any apparent change in the total level of 4.1. In addition, the analysis of the aged cell membranes did not show any large-molecular-mass aggregated protein at the origin of the SDS/polyacrylamide gels, indicating a lack of transglutaminase activity in the senescence process for rabbit erythrocytes. These results indicate that aging of the rabbit erythrocyte is not accompanied by gross proteolytic degradation or transglutaminase-catalysed cross-linking of membrane components.     

Sequences essential for IS50 transposition. The first base-pair

Sequences near the ends of the insertion element IS50 are essential for its transposition, probably because they serve as sites upon which the IS50-encoded transposase protein acts. To determine if these essential sequences include the first base-pair at each end of IS50 we generated 5'C to 5'G transversions at these positions. Each mutation reduced the transposition frequency to 1% to 2% of wild-type. DNA sequence analyses showed that the mutant 5'G is preserved during transposition.     

Fis plays a role in Tn5 and IS50 transposition.

The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.     

A sequence at the inside end of IS50 down regulates transposition.

Deletion analysis has shown that the segment at the IS50 inside (I) end that is needed for efficient transposition is approximately 19 bp long. Dam methylation at two 5' GATC sequences within this segment decreases I-end transposition activity. A third 5' GATC sequence is present at bp 21-24 of the I end. The comparisons presented here show that extension of the I end from 19 to 24 bp decreases its transposition activity in dam cells 5- to 50-fold, depending on the overall transposon structure.     

Abnormalities and reduced reproductive potential of sperm from Tnp1- and Tnp2-null double mutant mice

Previous studies have demonstrated the importance of transition nuclear proteins, TP1 and TP2, in spermatogenesis and male fertility. However, importance of the overall level of transition proteins and their level of redundancy in the production of normal sperm is not clear. Epididymal sperm from the nine possible Tnp1 and Tnp2 null genotypes demonstrated a general decrease in normal morphology, motility, chromatin condensation, and degree of protamine 2 processing with decreasing levels of transition proteins in mutant sperm. Nuclei of some mutant epididymal sperm stained poorly with hematoxylin and DNA fluorochromes, suggesting that the DNA of these sperm underwent degradation during epididymal transport. When epididymal sperm were injected directly into oocytes, fertilization and embryonic development were reduced only in the two most severely affected genotypes. These phenotypes indicated some functional redundancy of transition proteins; however, redundancy of transition protein function was not complete, as, for example, sperm from double heterozygous males had fewer abnormalities than sperm from males homozygous for a single Tnp null mutation. Our study suggests that each TP fulfills some unique function during spermiogenesis even though sperm phenotypes strongly indicate defects are largely attributable to an overall gene dosage effect. Similarities between sperm defects found in Tnp mutants and infertile patients make the Tnp mutants a valuable tool with which to study outcomes following fertilization using sperm with compromised DNA integrity.     

Membrane association of the Escherichia coli enterobactin synthase proteins EntB/G, EntE, and EntF

The cytosolic proteins EntE, EntF, and EntB/G, which are Escherichia coli enzymes necessary for the final stage of enterobactin synthesis, are released by osmotic shock. Here, consistent with the idea that cytoplasmic proteins found in shockates have an affinity for membranes, a small fraction of each was found in membrane preparations. Two procedures demonstrated that the enzymes were enriched in a minor membrane fraction of buoyant density intermediate between that of cytoplasmic and outer membranes, providing indirect support for the notion that these proteins have a role in enterobactin excretion as well as synthesis.     

Membrane proteins involved in pollen-pistil interactions.

Self-incompatibility (SI) is a widespread mechanism in angiosperms which prevents self-fertilization. This mechanism relies on cell-cell interactions between pollen and pistil. Among the different SI systems that have been reported, two have been particularly investigated: the gametophytic system of Solanaceae and the sporophytic system of Brassicaceae. In these two families, although the molecular bases of SI response are different, secreted and/or membrane-anchored proteins are required for self-pollen rejection. Interestingly, these proteins exhibit two functions: recognition and a catalytic activity. In this review article, we present recent advances which permit a better understanding of how these proteins control the male/female recognition event associated with the SI response.