Perfusion CT helps decision making for thrombolysis when there is no clear time of onset.

Current guidelines on thrombolysis post stroke with recombinant tissue plasminogen activator (rt-PA) exclude its use where time of onset is unknown, thus denying some patients potentially beneficial treatment. Contrast enhanced perfusion computed tomography (pCT) imaging can be used together with plain CT and information on clinical deficits to decide whether or not thrombolysis should be initiated even though the exact time of stroke onset is unknown. Based on the results of pCT and CT, rt-PA was administered to two patients with unknown time of stroke onset; one of the patients also underwent suction thrombectomy. Results in both cases were excellent.

Mu and IS1 transpositions exhibit strong orientation bias at the Escherichia coli bgl locus.

The region upstream of the Escherichia coli bgl operon is an insertion hot spot for several transposons. Elements as distantly related as Tn1, Tn5, and phage Mu home in on this location. To see what characteristics result in a high-affinity site for transposition, we compared in vivo and in vitro Mu transposition patterns near the bgl promoter. In vivo, Mu insertions were focused in two narrow zones of DNA near bgl, and both zones exhibited a striking orientation bias. Five hot spots upstream of the bgl cyclic AMP binding protein (CAP) binding site had Mu insertions exclusively with the phage oriented left to right relative to the direction of bgl transcription. One hot spot within the CAP binding domain had the opposite (right-to-left) orientation of phage insertion. The DNA segment lying between these two Mu hot-spot clusters is extremely A/T rich (80%) and is an efficient target for insertion sequences during stationary phase. IS1 insertions that activate the bgl operon resulted in a decrease in Mu insertions near the CAP binding site. Mu transposition in vitro differed significantly from the in vivo transposition pattern, having a new hot-spot cluster at the border of the A/T-rich segment. Transposon hot-spot behavior and orientation bias may relate to an asymmetry of transposon DNA-protein complexes and to interactions with proteins that produce transcriptionally silenced chromatin.

Transcription induces a supercoil domain barrier in bacteriophage Mu.

In enteric bacteria, chromosomes are partitioned into domains that exhibit restricted supercoil movement. The most common domain barrier detected by gammadelta resolution assays is random with respect to sequence and occurs more frequently in cells growing rapidly in rich medium compared to cells in stationary phase. Transcription generates both positive and negative supercoiling movement. To address the question of whether transcription causes the appearance of new domain boundaries, a transcriptionally active MudI element was substituted for a MudJr-1 element that resides within the cobT gene of Salmonella typhimurium. Mu-specific transcription from the phage early promoter was placed under control of either the wild type (c(+)) or the temperature-sensitive (cts62) repressor. Using a resolution assay with res sites at six chromosomal locations, domain structure was normal in cells carrying the MudAr-1 prophage with a wild type Mu repressor. However, in cells with a MudAr-1 prophage harboring the cts62 repressor, a new domain barrier appeared in > 90% of the cells. Supercoil movement was restricted ahead of but not behind the transcription machinery. We conclude that the strong Mu early promoter induces the appearance of a domain barrier within the limits of a MudAr-1 prophage.

Phage Mu transposition immunity reflects supercoil domain structure of the chromosome.

Transposition immunity is the negative influence that the presence of one transposon sequence has on the probability of a second identical element inserting in the same site or in sites nearby. A transposition-defective Mu derivative (MudJr1) produced transposition immunity in both directions from one insertion point in the Salmonella typhimurium chromosome. To control for the sequence preference of Mu transposition proteins, Tn10 elements were introduced as targets at various distances from an immunity-conferring MudJr1 element. Mu transposition into a Tn10 target was not detectable when the distance of separation from MudJr1 was 5 kb, and transposition was unencumbered when the separation was 25 kb. Between 5 kb and 25 kb, immunity decayed gradually with distance. Immunity decayed more sharply in a gyrase mutant than in a wild-type strain. We propose that Mu transposition immunity senses the domain structure of bacterial chromosomes.

Gyrase and Topo IV modulate chromosome domain size in vivo.

In bacteria, DNA supercoil movement is restricted to subchromosomal regions or 'domains.' To elucidate the nature of domain boundaries, we analysed reaction kinetics for gammadelta site-specific resolution in six chromosomal intervals ranging in size from 14 to 90 kb. In stationary cultures of Salmonella typhimurium, resolution kinetics were rapid for both short and long intervals, suggesting that random stationary barriers occur with a 30% probability at approximately 80 kb intervals along DNA. To test the biochemical nature of domain barriers, a genetic screen was used to look for mutants with small domains. Rare temperature-sensitive alleles of DNA gyrase and Topo IV (the two essential type II topoisomerases) had more supercoil barriers than wild-type strains in all growth states. The most severe gyrase mutants were found to have twice as many barriers in growing cells as wild type throughout a 90 kb interval of the chromosome. We propose that knots and tangles in duplex DNA restrain supercoil diffusion in living bacteria.

The DNA cleavage reaction of DNA gyrase. Comparison of stable ternary complexes formed with enoxacin and CcdB protein.

The potent synthetic fluoroquinolones and the natural CcdB protein encoded by the F plasmid both inhibit bacterial growth by attacking DNA gyrase and by stimulating enzyme-induced breaks in bacterial DNA. The cleavage mechanisms of these structurally diverse compounds were analyzed by purifying and characterizing stable ternary complexes of enoxacin and CcdB protein with gyrase bound to a strong gyrase binding site from bacteriophage Mu. Three differences between enoxacin- and CcdB-derived complexes were discovered. 1) Enoxacin binds to the DNA active site and alters the breakage/reunion activity of the enzyme. CcdB binds gyrase-DNA complexes but does not influence enzymatic activity directly. 2) Complexes that produce DNA cleavage with enoxacin are reversible, whereas similar complexes made with CcdB protein are not. 3) Enoxacin stimulates cleavage of both relaxed and supercoiled forms of DNA in the absence of ATP, whereas CcdB induces cleavage only after many cycles of ATP-dependent breakage and reunion. These differences in mechanisms can be explained by a model in which enoxacin induces formation of a novel "cleavable" complex, whereas CcdB protein traps a very rare "cleaved" conformation of the enzyme.

Surveying a supercoil domain by using the gamma delta resolution system in Salmonella typhimurium.

A genetic system was developed to investigate the supercoil structure of bacterial chromosomes. New res-carrying transposons were derived from MudI1734 (MudJr1 and MudJr2) and Tn10 (Tn10dGn). The MudJr1 and MudJr2 elements each have a res site in opposite orientation so that when paired with a Tn10dGn element in the same chromosome, one MudJr res site will be ordered as a direct repeat. Deletion formation was studied in a nonessential region (approximately 100 kb) that extends from the his operon through the cob operon. Strains with a MudJr insertion in the cobT gene at the 5' end of the cob operon plus a Tn10dGn insertion positioned either clockwise or counterclockwise from cobT were exposed to a burst of RES protein. Following a pulse of resolvase expression, deletion formation was monitored by scoring the loss of the Lac+ phenotype or by loss of tetracycline resistance. In exponentially growing populations, deletion products appeared quickly in some cells (in 10 min) but also occurred more than an hour after RES induction. The frequency of deletion (y) diminished with increasing distance (x) between res sites. Results from 15 deletion intervals fit the exponential equation y = 120 . 10(-0.02x). We found that res sites can be plectonemically interwound over long distances ( > 100 kb) and that barriers to supercoil diffusion are placed stochastically within the 43- to 45-min region of the chromosome.

C-terminal deletions can suppress temperature-sensitive mutations and change dominance in the phage Mu repressor.

Mutations in an N-terminal 70-amino acid domain of bacteriophage Mu's repressor cause temperature-sensitive DNA-binding activity. Surprisingly, amber mutations can conditionally correct the heat-sensitive defect in three mutant forms of the repressor gene, cts25 (D43-G), cts62 (R47-Q) and cts71 (M28-I), and in the appropriate bacterial host produce a heat-stable Sts phenotype (for survival of temperature shifts). Sts repressor mutants are heat sensitive when in supE or supF hosts and heat resistant when in Sup degrees hosts. Mutants with an Sts phenotype have amber mutations at one of three codons, Q179, Q187, or Q190. The Sts phenotype relates to the repressor size: in Sup degrees hosts sts repressors are shorter by seven, 10, or 18 amino acids compared to repressors in supE or supF hosts. The truncated form of the sts62-1 repressor, which lacks 18 residues (Q179-V196), binds Mu operator DNA more stably at 42 degrees in vitro compared to its full-length counterpart (cts62 repressor). In addition to influencing temperature sensitivity, the C-terminus appears to control the susceptibility to in vivo Clp proteolysis by influencing the multimeric structure of repressor.

End joining of genomic DNA and transgene DNA in fertilized mouse eggs.

A linear 5.2-kb HS2/beta-globin construct with an upstream KpnI terminus (4-nucleotide (nt) 3' protruding single strand, PSS) and a downstream SalI terminus (4-nt 5' PSS) was microinjected into fertilized mouse eggs. The injected DNA fragments integrated into the mouse genome primarily as a head-to-tail tandem array. Chromosome/transgene junctions were obtained from seven of eight transgenic animals. All of the junctions occurred in the proximity of a transgene KpnI end; a maximum loss of 8 nt from the transgene terminus was observed. Two of these junctions completely preserved the 4-nt KpnI 3' PSS. Transgene/transgene junctions from two animals were analyzed. SalI/KpnI junctions that completely preserved both the SalI 5' PSS and the KpnI 3' PSS were found in each animal. These are the first examples of complete nt preservation at junctions formed between a 5' PSS terminus and a 3' PSS terminus in transgenic mice. The data are consistent with the fill-in model of Thode et al. [Cell 60 (1990) 921-928] in which alignment proteins juxtapose 5' PSS and 3' PSS termini; DNA polymerase then utilizes the recessed 3'-OH of the 5' PSS terminus as a primer to synthesize DNA across the gap. This mechanism results in the formation of junctions with no loss of sequence. The results described in the present paper suggest that this mechanism may be involved in the formation of junctions in transgenic mice.

Characterization of Mu prophage lacking the central strong gyrase binding site: localization of the block in replication.

Bacteriophage Mu contains an unusually strong DNA gyrase binding site (SGS), located near the center of its genome, that is required for efficient Mu DNA replication (M. L. Pato, Proc. Natl. Acad. Sci. USA 91:7056-7060, 1994; M. L. Pato, M. M. Howe, and N. P. Higgins, Proc. Natl. Acad. Sci. USA 87:8716-8720, 1990). Replication of wild-type Mu initiates about 10 min after induction of a lysogen, while replication in the absence of the SGS is delayed about an hour. To determine which step in the replication pathway is blocked in the absence of the SGS, we inactivated the SGS by deletion and by insertion and studied the effects of these alterations on various stages of Mu DNA replication. Following induction in the absence of a functional SGS, early transcription and synthesis of the Mu-encoded replication proteins occurred normally. However, neither strand transfer nor cleavage at the Mu genome termini could be detected 40 min after induction. The data are most consistent with a requirement for the SGS in the efficient synapsis of the Mu prophage termini to form a separate chromosomal domain.

'Muprints' of the lac operon demonstrate physiological control over the randomness of in vivo transposition.

A method called Muprinting has been developed that uses PCR to generate a detailed picture of the bacteriophage Mu transposition sites in chosen domains of the bacterial chromosome. Muprinting experiments in Escherichia coli show that the frequency of phage integration changes dramatically near two repressor binding sites in the lac operon. When the lac operon was repressed, hotspots for Mu transposition were found near the O1 and O2 operators that are proposed to make a repression loop. When cells were grown in lactose, Mu transposition near these operators was greatly diminished. Striking changes in transposition frequencies were limited to the control region and were not found in a region of the lacZ gene lying beyond the O2 operator. Muprints of the bgl operon showed a different pattern; hotspots for Mu transposition detected in sequences upstream of the bglC promoter when the operon was silenced changed when the operon became activated by mutation. By targeting transposition to the regulatory regions around non-expressed genes, Mu may demonstrate a self-restraint mechanism that allows the virus to move through its host genome without disrupting the functions that contribute to a healthy cell physiology.

H-NS over-expression induces an artificial stationary phase by silencing global transcription.

Bacteria organize their chromosomes in a complex interwound supercoiled structure called the nucleoid through the action of topoisomerases and a set of small (10-20 kDa) proteins. The two most abundant nucleoid-associated proteins are HU and H-NS. H-NS increases in abundance during stationary phase. Over-expression of HU is well tolerated and compatible with transcription and cell growth. Increasing the concentration of H-NS leads to a rapid silencing of global transcription and produces a growth-arrested state reminiscent of stationary phase. H-NS over-expression also induces a substantial loss of supercoiling in plasmid DNA during the time that transcription is arrested. Comparing the effects of over-expression of these two proteins gives some insight into the differential roles of these proteins in the activity of the chromosome. These observations are interpreted in a model of nucleoid organization.

Expression of the gene encoding the major bacterial nucleotide protein H-NS is subject to transcriptional auto-repression.

Expression of a promoterless cat gene fused to a DNA fragment of approximately 400 bp, beginning at -313 of Escherichia coli hns, was significantly repressed in E. coli and Salmonella typhimurium strains with wild-type hns but not in mutants carrying hns alleles. CAT expression from fusions containing a shorter (110 bp) segment of hns was essentially unaffected in the same genetic backgrounds. The stage of growth was found to influence the extent of repression which was maximum (approximately 75%) in mid-log cultures and negligible in cells entering the stationary phase. The level of repression in early-log phase was lower than in mid-log phase cultures, probably because of the presence of high levels of Fis protein, which counteracts the H-NS inhibition by stimulating hns transcription. The effects observed in vivo were mirrored by similar results obtained in vitro upon addition of purified H-NS and Fis protein to transcriptional systems programmed with the same hns-cat fusions. Electrophoretic gel shift assays, DNase I footprinting and cyclic permutation gel analyses revealed that H-NS binds preferentially to the upstream region of its own gene recognizing two rather extended segments of DNA on both sides of a bend centred around -150. When these sites are filled by H-NS, an additional site between approximately -20 and -65, which partly overlaps the promoter, is also occupied. Binding of H-NS to this site is probably the ultimate cause of transcriptional auto-repression.

Stabilization of bacteriophage Mu repressor-operator complexes by the Escherichia coli integration host factor protein.

All of the previously described effects of integration host factor (IHF) on bacteriophage Mu development have supported the view that IHF favours transposition-replication over the alternative state of lysogenic phage growth. In this report we show that, consistent with a model in which Mu repressor binding to its operators requires a particular topology of the operator DNA, IHF stimulates repressor binding to the O1 and O2 operators and enhances Mu repression. IHF would thus be one of the keys, besides supercoiling and the H-NS protein, that lock the operator region into the appropriate topological conformation for high-affinity binding not only of the phage transposase but also of the phage repressor.

Death and transfiguration among bacteria.

When bacteria are placed in sub-optimal environments, they can respond by increasing the frequency of mutants created by base substitution, frame-shift and transposition mutations. Also, during periods of restrictive growth, 'dead' bacterial cells may transfer genetic material to neighboring colony-forming cells. This can be beneficial, resulting in a heterogeneous population that may exhibit differentiation and even produce killer cells. These discoveries reveal several conundrums about the control of an organism over mutations and the supposed randomness of genetic variation.

Temperature-sensitive mutations in the bacteriophage Mu c repressor locate a 63-amino-acid DNA-binding domain.

Phage Mu's c gene product is a cooperative regulatory protein that binds to a large, complex, tripartite 184-bp operator. To probe the mechanism of repressor action, we isolated and characterized 13 phage mutants that cause Mu to undergo lytic development when cells are shifted from 30 to 42 degrees C. This collection contained only four mutations in the repressor gene, and all were clustered near the N terminus. The cts62 substitution of R47----Q caused weakened specific DNA recognition and altered cooperativity in vitro. A functional repressor with only 63 amino acids of Mu repressor fused to a C-terminal fragment of beta-galactosidase was constructed. This chimeric protein was an efficient repressor, as it bound specifically to Mu operator DNA in vitro and its expression conferred Mu immunity in vivo. A DNA looping model is proposed to explain regulation of the tripartite operator site and the highly cooperative nature of repressor binding.

Frameshift mutations in the bacteriophage Mu repressor gene can confer a trans-dominant virulent phenotype to the phage.

Virulent mutations in the bacteriophage Mu repressor gene were isolated and characterized. Recombination and DNA sequence analysis have revealed that virulence is due to unusual frameshift mutations which change several C-terminal amino acids. The vir mutations are in the same repressor region as the sts amber mutations which, by eliminating several C-terminal amino acids, suppress thermosensitivity of repressor binding to the operators by its N-terminal domain (J. L. Vogel, N. P. Higgins, L. Desmet, V. Geuskens, and A. Toussaint, unpublished data). Vir repressors bind Mu operators very poorly. Thus the Mu repressor C terminus, either by itself or in conjunction with other phage or host proteins, tunes the DNA-binding properties at the repressor N terminus.

Mutations altering chromosomal protein H-NS induce mini-Mu transposition.

Bacteriophage Mu is one of the most efficient transposons known, capable of moving a hundred viral copies to new positions in the bacterial chromosome in an hour. Mu also forms stable lysogens. In bacteria lysogenic for the defective protein fusion-forming phage MudII1681, which can transpose and replicate but does not encode genes for DNA packaging and cell lysis, the frequency of transposition changes as colonies age. To find host genes that alter the spontaneous Mu transposition frequency, we used a genetic screen with mini-MudlacZ fusion formation as an assay. H-NS (also called H1a and B1) is an abundant nonspecific DNA-binding protein localized to the bacterial chromosome. H-NS has an unusual structure of interspersed patches of acidic and basic residues reminiscent of eukaryotic HMG proteins. Mutations in hns caused an increase in Mu-specific transcription and a dramatic increase in MudII1681 transposition rates when cells were put under certain growth conditions. Purified H-NS stabilized Mu repressor-DNA complexes in vitro, suggesting that H-NS contributes to the organization of transcriptionally inactive DNA in vivo.

Topoisomerase mutants and physiological conditions control supercoiling and Z-DNA formation in vivo.

The influence of topoisomerase I and gyrase mutations in Escherichia coli on the supercoiled density of recombinant plasmids and the stability of left-handed Z-DNA was investigated. The formation of Z-DNA in vivo by dC-dG sequences of different lengths was used to determine the effective plasmid supercoil densities in the mutant strains. The presence of Z-DNA in the cells was detected by linking number and EcoRI methylase inhibition assays. A change in the unrestrained superhelical tension in vivo directly effects the B- to Z-DNA transition. Alterations in the internal or external environment of the cells, such as the inactivation of gyrase or topoisomerase I, a gyrase temperature-sensitive mutant, or starvation of cells, have a dramatic influence on the topology of plasmids. Also, E. coli has significantly more superhelical strain than Klebsiella, Morganella, or Enterobacter. These studies indicate that linking deficiency and effective supercoil density are mutually independent variables of plasmid tertiary structure. A variety of factors, such as protein-DNA interactions, activity of topoisomerases, and the resulting supercoil density, contribute to the B to Z transition inside living cells.

A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition.

We have discovered a centrally located site that is required for efficient replication of bacteriophage Mu DNA and identified it as a strong DNA gyrase-binding site. Incubation of Mu DNA with gyrase and enoxacin revealed a cleavage site 18.1 kilobases from the left end of the 37.2-kilobase genome. Two observations indicate a role for the site in Mu replication: mutants of Mu, able to grow on an Escherichia coli gyrB host that does not allow growth of wild-type Mu, were found to possess single-base changes resulting in more efficient gyrase binding and cleavage at the site. Introduction of a 147-base-pair deletion that eliminated the site from a prophage inhibited the onset of Mu replication for greater than 1 hr after induction.