Interference between M13 and oriM13 plasmids is mediated by a replication enhancer sequence near the viral strand origin

The origin of replication for the viral strand of bacteriophage M13 DNA is contained within a 507 base-pair intergenic region of the phage chromosome. The viral strand origin is defined as the specific site at which the M13 gene II protein nicks the duplex replicative form of M13 DNA to initiate rolling-circle synthesis of progeny viral DNA. Using in vitro techniques we have constructed deletion mutations in M13 DNA at the unique AvaI site which is located 45 nucleotides away on the 3' side of the gene II protein nicking site. This deletion analysis has identified a sequence near the viral strand origin that is required for efficient replication of the M13 genome. We refer to this part of the intergenic region as a "replication enhancer" sequence. We have also studied the function of this sequence in chimeric pBR322-M13 plasmids and found that plasmids carrying both the viral strand origin and the replication enhancer sequence interfere with M13 phage replication. Based upon these findings we propose a model for the mechanism of action of the replication enhancer sequence involving binding of the M13 gene II protein.     

Deletion analysis of the cloned replication origin region from bacteriophage M13.

A cloned 270-nucleotide fragment from the origin region of the M13 duplex replicative form DNA confers an M13-dependent replication mechanism upon the plasmid vector pBR322. This M13 insert permits M13 helper-dependent replication of the hybrid plasmid in polA cells which are unable to replicate the pBR322 replicon alone. Using in vitro techniques, we have constructed several plasmids containing deletions in the M13 DNa insert. The endpoints of these deletions have been determined by DNA sequence analysis and correlated with the transformation and replication properties of each plasmid. Characterization of these deletion plasmids allows the following conclusions. (i) The initiation site for M13 viral strand replication is required for helper-dependent propagation of the chimeric plasmid. (ii) A DNA sequence in the M13 insert, localized between 89 and 129 nucleotides from the viral strand initiation site, is necessary for efficient transformation of polA cells. A chimeric plasmid containing the viral strand initiation site, but lacking this additional 40 nucleotide M13 sequence, transforms helper-infected cells at a frequency approximately 10(4)-fold less than that of plasmids containing this additional DNA segment. (iii) The entire M13 complementary strand origin can be deleted without affecting M13-dependent transformation by the hybrid plasmids. We propose a model in which replication of one strand of duplex chimera initiates by nicking at the gene II protein nicking site in the viral strand of the M13 insert, followed by asymmetric single-strand synthesis. Initiation of the complementary strand possibly occurs within plasmid sequences.     

Characterization of the genome of the mealybug Planococcus lilacinus,a model organism for studying whole-chromosome imprinting and inactivation

The co-occurrence of three chromosome-wide phenomena--imprinting, facultative heterochromatization and diffuse centromere--in the mealybug Planococcus lilacinus makes investigation of the genomics of this species an attractive prospect. In order to estimate the complexity of the genome of this species, 300 random stretches of its DNA, constituting approximately 0.1% of the genome, were sequenced. Coding sequences appear to constitute approximately 53.5%, repeat sequences approximately 44.5% and non-coding single-copy sequences approximately 2% of the genome. The proportion of repetitive sequences in the mealybug is higher than that in the fruit fly Drosophila melanogaster (approximately 30%). The mealybug genome (approximately 220 Mb) is about 1.3 times the size of the fly genome (approximately 165 Mb) and its GC content (approximately 35%) less than that of the fly genome (approximately 40%). The relative abundance of various dinucleotides, as analysed by the method of Gentles and Karlin, shows that the dinucleotide signatures of the two species are moderately similar and that in the mealybug there is neither over-representation nor under-representation of any dinucleotide.     

Non lethal concentrations of pesticide impair ovarian function in the freshwater perch,Anabas testudineus

Synopsis Anabas testudineus were treated with non lethal levels of metacid-50 (0.106 ppb) and carbaryl (1.66 ppm) for 90 days covering the pre-spawning and spawning phases of the annual reproductive cycle. The main purpose of the present work was to identify the effects of metacid-50 and carbaryl on the gonado somatic index (GSI) and ovarian and plasma estrogen level. There was no alteration in GSI until 15 days, initiating the inhibition on day 20 which further intensified from 20 to 90 days of exposure. Plasma and ovarian estrogen level significantly increased up to 15 days of exposure followed by a decline till the end of the experiment. It is noteworthy that the effect of pesticides on GSI is reflected in the ovarian estrogen level. This highlights the fact that at short-term exposures the nonlethal levels of pesticides have no inhibitory effect while at long-term exposure, the pesticides have potent inhibitory effect on the reproduction of fish.     

Cardiac output of marmots by the thoracic impedance technique.

Baseline impedance (Z₀) and resistivity of blood were higher for marmots than reported for other species. The transthoracic impedance method was compared to dye dilution and electromagnetic flowmeter procedures for estimation of cardiac output of seven marmots at a range of flows from 40 to 400 ml/min. There was a low, positive, but significant correlation (r = 0.566) found in comparison to dye dilution at outputs measured by the impedance method exceeding 120 ml/min. Correlation was better (r = 0.905) in the comparison between impedance and flowmeter methods.It was concluded that transthoracic impedance provides data that are sufficiently accurate for chronic measurements of stroke volume and cardiac output of this species. The method has the additional advantage of supplying EGG and respiratory data without supplemental connections to the animal preparations.     

Partial suppression of an Escherichia coli TonB transmembrane domain mutation (ΔV17) by a missense mutation in ExbB

Active transport of vitamin B₁₂ and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the amino-terminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The ΔV17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonBΔV17. Molecular modeling suggested that the ΔV17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonBΔV17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the ΔV17 mutation interrupted productive TonB–ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonBΔV17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonBΔV17 export. Unlike the tonBΔV17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonBδV17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (≅ 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.     

Development and neuromodulation of spinal locomotor networks in the metamorphosing frog

Metamorphosis in the anuran frog, Xenopus laevis, involves profound structural and functional transformations in most of the organism's physiological systems as it encounters a complete alteration in body plan, habitat, mode of respiration and diet. The metamorphic process also involves a transition in locomotory strategy from axial-based undulatory swimming using alternating contractions of left and right trunk muscles, to bilaterally-synchronous kicking of the newly developed hindlimbs in the young adult. At critical stages during this behavioural switch, functional larval and adult locomotor systems co-exist in the same animal, implying a progressive and dynamic reconfiguration of underlying spinal circuitry and neuronal properties as limbs are added and the tail regresses. To elucidate the neurobiological basis of this developmental process, we use electrophysiological, pharmacological and neuroanatomical approaches to study isolated in vitro brain stem/spinal cord preparations at different metamorphic stages. Our data show that the emergence of secondary limb motor circuitry, as it supersedes the primary larval network, spans a developmental period when limb circuitry is present but not functional, functional but co-opted into the axial network, functionally separable from the axial network, and ultimately alone after axial circuitry disappears with tail resorption. Furthermore, recent experiments on spontaneously active in vitro preparations from intermediate metamorphic stage animals have revealed that the biogenic amines serotonin (5-HT) and noradrenaline (NA) exert short-term adaptive control over circuit activity and inter-network coordination: whereas bath-applied 5-HT couples axial and appendicular rhythms into a single unified pattern, NA has an opposite decoupling effect. Moreover, the progressive and region-specific appearance of spinal cord neurons that contain another neuromodulator, nitric oxide (NO), suggests it plays a role in the maturation of limb locomotor circuitry. In summary, during Xenopus metamorphosis the network responsible for limb movements is progressively segregated from an axial precursor, and supra- and intra-spinal modulatory inputs are likely to play crucial roles in both its functional flexibility and maturation.     

31P NMR studies of enzyme-bound substrate complexes of yeast 3-phosphoglycerate kinase: III. Two ADP binding sites and their Mg(II) affinity; effects of vanadate and arsenate on enzymic complexes with ADP and 3-P-glycerate

31P nuclear magnetic resonance (NMR) measurements (at 121.5 MHz and 5 degrees C) were made on complexes of 3-phosphoglycerate kinase with ADP and 3-P-glycerate. Addition of Mg(II) to E.ADP shifts the alpha-P signal downfield by 3.8 ppm such that the alpha-P signal superimposes that for beta-P(E.MgADP). Such a shift is atypical among the Mg(II)-nucleotide complexes with other ATP-utilizing enzymes. This shift allowed the determination that enzyme bound ADP is saturated with Mg(II) for [Mg(II)]/[ADP] = 3.0--similar to that reported for ATP complexes with this enzyme (B.D. Ray and B.D. Nageswara Rao, Biochemistry 27, 5574 (1988]. This parallel behavior suggests that ADP binds at two sites on the enzyme as does ATP with disparate Mg(II) affinities. 31P relaxation times in E.MnADP.vanadate.3-P-glycerate and E.CoADP.vanadate.3-P-glycerate complexes indicate that these are long-lived, tightly bound complexes. 31P chemical shift measurements on diamagnetic complexes (with Mg(II] revealed three signals in the 2-5 ppm region (attributable to 3-P-glycerate) only upon addition of all the components necessary to form the E.MgADP.vanadate.3-P-glycerate complex. Subsequent sequestration of Mg(II) from the complex with excess EDTA reversed the Mg(II) induced effects on the ADP signals but did not cause coalescence of the three signals seen in the 2-5 ppm region. Addition of excess sulfate to dissociate these complexes from the enzyme resulted in a single resonance of 3-P-glycerate. The use of arsenate in place of vanadate yielded very similar results. These results suggest that, in the presence of MgADP, vanadate or arsenate, and 3-P-glycerate, the enzyme catalyzed the formation of multiple structurally distinguishable complexes that are stable on the enzyme and labile off the enzyme.