The regulatory role of the IS 1-encoded InsA protein in transposition

We show here that the protein InsA, which is encoded by IS 1 and binds specifically to the terminal inverted repeats of this insertion sequence, negatively regulates IS 1 transposition activity. We demonstrate that it inhibits both IS 1-mediated cointegrate formation and transposition of a synthetic IS 1-based transposon (‘omegon’Ω-on). These results also indicate that the Ω-on which does not itself encode IS 1 transposition functions can be complemented in trans, presumably by the copies of IS 1 resident in the Escherichia coli chromosome. Using insA-lacZ gene fusions, we show that at least part of this effect can be explained by the ability of InsA to repress expression of IS 1-encoded genes both in cis or in trans. The experiments involving Ω-on transposition raise the possibility that InsA inhibits transposition directly by competition with the transposase for their cognate site within the ends of IS 1.     

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.     

Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2.

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.     

Functional organization of the ends of IS 1: specific binding site for an IS1-encoded protein

The IS 1-encoded protein InsA binds specifically to both ends of IS1, and acts as a repressor of IS1 gene expression and may be a direct inhibitor of the transposition process. We show here, using DNasel 'foot-printing' and gel retardation, that the InsA binding sites are located within the 24/25 bp minimal active ends of IS1 and that InsA induces DNA bending upon binding. Conformational modification of the ends of IS1 as a result of binding of the host protein integration host factor (IHF) to its site within the minimal ends has been previously observed. Using a collection of synthetic mutant ends we have mapped some of the nucleotide sequence requirements for InsA binding and for transposition activity. We show that sequences necessary for InsA binding are also essential for transposition activity. We demonstrate that InsA and IHF binding sites overlap since some sequence determinants are shared by both InsA and IHF. The data suggest that these ends contain two functional domains: one for binding of InsA and IHF, and the other for transposition activity. A third region, when present, may enhance transposition activity with an intact right end. This 'architecture' of the ends of IS1 is remarkably similar to that of IS elements IS10, IS50 and IS903.     

Analysis of adenovirus early region 4-encoded polypeptides synthesized in productively infected cells.

Peptide-specific antisera were developed to analyze the products encoded by adenovirus type 5 early region 4 (E4) open reading frames 6 and 7. Reading frame 6 previously was shown to encode a 34-kilodalton polypeptide (34K polypeptide) that forms a complex with the early region 1B (E1B)-55K antigen and is required for efficient viral growth in lytic infection. Antisera that were generated recognized the E4-34K protein as well as a family of related polypeptides generated by the fusion of open reading frames 6 and 7. These polypeptides shared amino-terminal sequences with the 34K protein. Short-pulse analysis suggested that the heterogeneity observed with the 6/7 fusion products resulted from differential splicing patterns of related E4 mRNAs. An antiserum directed against the amino terminus of reading frame 6 recognized only the free form of the 34K antigen that was not associated with the E1B-55K protein. This observation allowed the determination of the stability of the free and complexed form of this polypeptide. Pulse-chase analyses demonstrated that both forms of the 34K protein had half-lives greater than 24 h, suggesting that complex formation did not result in stabilization of this gene product. The half-lives of the 6/7 fusion products were approximately 4 h. The 34K protein also was shown to have a nuclear localization within infected cells. Finally, analysis of a mutant carrying deletions in both the E4-34K and E1B-55K polypeptides indicated that the complex formed between these two proteins was a functional unit in lytic infection.     

Retinol-binding protein is synthesized in the mammalian eye

As the chromophoric component of the visual pigment, retinol plays an essential role in vision. In the plasma, retinol is transported by retinol-binding protein (RBP) in complex with transthyretin (TTR, prealbumin). In previous work we demonstrated intraocular synthesis of TTR. To determine whether RBP is also synthesized in the eye, we performed Northern and Western blot analysis of rat eye, and detected both RBP mRNA and immunoreactive RBP. Regional Northern analysis of bovine eye localized RBP mRNA to ciliary body/iris and retina/RPE. Preliminary immunohistochemical studies revealed a widespread but heterogeneous distribution of RBP in rat eye. We postulate that ocular RBP and TTR are involved in the intraocular translocation of retinol.     

Reduction of a 4q35-encoded nuclear envelope protein in muscle differentiation

Muscular dystrophy and peripheral neuropathy have been linked to mutations in genes encoding nuclear envelope proteins; however, the molecular mechanisms underlying these disorders remain unresolved. Nuclear envelope protein p19A is a protein of unknown function encoded by a gene at chromosome 4q35. p19A levels are significantly reduced in human muscle as cells differentiate from myoblasts to myotubes; however, its levels are not similarly reduced in all differentiation systems tested. Because 4q35 has been linked to facioscapulohumeral muscular dystrophy (FSHD) and some adjacent genes are reportedly misregulated in the disorder, levels of p19A were analyzed in muscle samples from patients with FSHD. Although p19A was increased in most cases, an absolute correlation was not observed. Nonetheless, p19A downregulation in normal muscle differentiation suggests that in the cases where its gene is inappropriately re-activated it could affect muscle differentiation and contribute to disease pathology.     

Polypeptides encoded by the early region of bacteriophage Mu synthesized in minicells of Escherichia coli

The preferential interaction of calf brain tubulin with glycerol in an aqueous buffer (0.01 m-NaP_i, 0.02 m-NaCl, 10^?4m-GTP, pH 7.0) has been investigated by densimetry. The apparent specific volumes of tubulin at constant chemical potential of the diffusible components were determined at 0, 10, 20 and 30% ($\text{v}\text{v}$) glycerol. Application of multicomponent solution thermodynamics shows that tubulin is preferentially hydrated in aqueous glycerol solvent and that such interaction results in thermodynamic destabilization of the system by raising the chemical potentials of both glycerol and tubulin. Interpreted in terms of the Wyman linkage function, the unfavorable free energy change brought about by the preferential protein-glycerol interaction can account for the glycerol enhancement of tubulin self-assembly in vitro into microtubules as well as offer a rationale for glycerol stabilization of the native tubulin conformation.     

Efficiency of phage protein synthesis in lambda-infected E. coli minicells

Phage lambda major head protein, the gene E product, has been identified among other phage proteins synthesized in lambda-infected Escherichia coli minicells, separated by SDS-acrylamide gel electrophoresis. On stained gels, the same protein has also been detected among total (bacterial and phage) proteins of lambda-infected minicells. The contribution of lambda proteins to the total protein content of lambda-infected minicells was found to be about 12% following 30 min lambda-infection. The inhibition of lambda early protein synthesis (shown by other authors in nucleate bacterial cells) practically does not occur in minicells; this may be the reason of the observed high efficiency of phage protein synthesis.     

Bacillus subtilis bacteriophage SPP1-encoded gene 34.1 product is a recombination-dependent DNA replication protein

SPP1-encoded replication and recombination proteins, involved in the early steps of the initiation of concatemeric DNA synthesis, have been analyzed. Dimeric G34.1P exonuclease degrades, with a 5' to 3' polarity and in a Mg2+-dependent reaction, preferentially linear double-stranded (ds) DNA rather than single-stranded (ss) DNA. Binding of the replisome organizer, G38P, to its cognate sites (oriDNA) halts the 5' to 3' exonucleolytic activity of G34.1P on dsDNA. The G35P recombinase increases the affinity of G34.1P for dsDNA, and stimulates G34.1P activity on dsDNA, but not on ssDNA. Then, filamented G35P promotes limited strand exchange with a homologous sequence. The ssDNA binding protein, G36P, protects ssDNA from the G34.1P exonuclease activity and stimulates G35P-catalyzed strand exchange. The data presented suggest a model for the role of G34.1P during initiation of sigma replication: G38P bound to oriDNA might halt replication fork progression, and G35P, G34.1P and G36P in concert might lead to the re-establishment of a unidirectional recombination-dependent replication that accounts for the direction of DNA packaging.     

Intestinal satiety protein apolipoprotein AIV is synthesized and regulated in rat hypothalamus

Apolipoprotein AIV (apo AIV) is a satiety protein secreted by the small intestine. We demonstrate for the first time that apo AIV protein and apo AIV mRNA are present in rat hypothalamus, a site intimately involved in the integration of signals for regulation of food intake and energy metabolism. We further characterized the regulation of hypothalamic apo AIV mRNA levels. Food-deprived animals showed a pronounced decrease in gene expression of apo AIV in the hypothalamus, with a concomitant decrease in the jejunum. Refeeding fasted rats with standard laboratory chow for 4 h evokes a significant increase of apo AIV mRNA in jejunum but not in hypothalamus. However, lipid refeeding to the fasted animals restored apo AIV mRNA levels both in hypothalamus and jejunum. Intracerebroventricular administration of apo AIV antiserum not only stimulated feeding, but also decreased apo AIV mRNA level in the hypothalamus. These data further confirm the central role of apo AIV in the regulation of food intake.     

phi X174-directed DNA and protein syntheses in infected minicells.

Phi X174-infected minicells, produced by Escherichia coli PC2251, synthesized 11 phi X174-encoded polypeptides. The infecting single-stranded viral genome was converted to a double-stranded, closed circular, replicative form (replicative form I). Little, if any, replicative form I replication took place, and synthesis of progeny single-stranded molecules could not be detected.     

Transferrin is a major mouse milk protein and is synthesized by mammary epithelial cells

Summary We have identified a major mouse milk protein as transferrin (Tf) using immunoprecipitation, 2-dimensional electrophoresis, Ouchterlony diffusion and V-8 protease digests. We show that Tf is synthesized by mammary epithelial cells themselves and that its synthesis and secretion is regulated distinctly from that of other milk proteins. In culture, the kinetics of Tf synthesis and secretion are distinct from that of β-casein; furthermore, Tf is relatively insensitive to lactogenic hormones whereas β-casein is hormone-dependent.In vivo, however, Tf is regulated by pregnancy. While the virgin gland produces small amounts of Tf, its production is greatly increased during pregnancy and lactation. Thus, Tf synthesis in the mammary gland is modulated by as yet unknown factorsin vivo. These observations are discussed in terms of Tf’s possible role in mammary gland growth, differentiation and function. This research was supported by the OHER office of U. S. DOE, contract DE-AC 03-76S F00098, and NIH grant BRSG RR05918. Editor’s Statement This study combines cultured cells and direct analytical approaches to show that authentic transferrin is a major mouse milk protein and is regulated differently than beta-casein in mammary epithelium. Wallace L. McKeehan     

Human metallothionein-II is synthesized as a stable membrane-localized fusion protein in Escherichia coli

A synthetic gene encoding human metallothionein-II (HMT) was cloned into the specially constructed high-copy-number expression vector, pUA7, and expressed in Escherichia coli. The plasmid construct includes the promoter/operator and regulatory sequences of the Salmonella typhimurium ara operon and part of the 5'-coding and all of the 3'-noncoding regions of the E. coli lpp. Upon induction with arabinose, the resulting Lpp::HMT fusion protein was produced 75,000-fold over uninduced cells, with a relatively stable mRNA (T1/2 of 8.3 min) and a completely stable protein. In addition, over 95% of the final fusion protein was localized in the outer membrane and was capable of binding heavy metals (especially cadmium) in vitro. Cells producing Lpp::HMT bioaccumulated heavy metals (e.g., cadmium) 66-fold over nonproducing cells.     

Identification of the repressor and repressor bypass (antirepressor) polypeptides of bacteriophage P1 synthesized in infected minicells

Summary P1 infected minicells synthesize approximately 50 phage-encoded polypeptides. Phage expression is temporally controlled, demonstrating phage polypeptides synthesized both early and late after infection. The P1 repressor, gpc1 ¹ (Mr=33,000), repressor bypass polypeptide, gprebA (Mr=27,500) and cistron 10 product, (gp10) (Mr=64,000), have been identified by infection of minicells with P1 amber mutants. The beta-lactamase gene product (gpbla) carried by the closely related phage P7 and the chloramphenicol acetyl-transferase gene product (gpcat) carried by P1 Cm (in Tn9) have been demonstrated. Infection of minicells by P1vir ^s or P1c4 mutants results in increased synthesis of gprebA and a second polypeptide designated gprebB (Mr=40,000). The P1vir11 mutation leads to increased synthesis of a small polypeptide (Mr=3,500) but does not affect the amount of gpc1 synthesized.