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The phase diagram of a hard-sphere fluid in the presence of a random pinning potential is studied analytically and numerically. In the analytic work, replicas are introduced for averaging over the quenched disorder, and the hypernetted chain approximation is used to calculate density correlations in the replicated liquid. The freezing transition of the liquid into a nearly crystalline state is studied using a density-functional approach, and the liquid to glass transition is studied using a phenomenological replica symmetry breaking approach. In the numerical work, local minima of a discretized version of the Ramakrishnan-Yussouff free-energy functional are located and the phase diagram in the density-disorder plane is obtained from an analysis of the relative stability of these minima. Both approaches lead to similar results for the phase diagram. The first-order liquid to crystalline solid transition is found to change to a continuous liquid to glass transition as the strength of the disorder is increased above a threshold value.

The accords reached at Bonn and Marrakesh resolved important operational questions left open at Kyoto and gave final shape to the Kyoto Protocol. This achievement came at a considerable price in terms of defining the Kyoto targets as well as procedural transparency. The accords have drained ‘supplementarity’ of all operational significance. It imposes no ceiling on the extent to which an Annex I Party may take recourse to the ‘flexibility mechanisms’. Thus over 50% of the notional reduction in emissions is likely to be accounted for by trading in ‘hot air’—a mere accounting device. This figure could be even larger unless steps are taken to ensure that the entry of Kazakhstan (and any other country) into Annex I for the purposes of the protocol does not lead to a further expansion in the supply of ‘hot air’. The agreed ceilings for additions to and subtractions from the assigned amounts of Parties for forest management activities will enable the Annex I Parties to account for another 38.31% of their notional emission reduction commitments. There was a certain lack of transparency in the manner in which the ceilings were calculated. Thus the actual reduction in emissions required of the Annex I Parties will be very modest—just over 11% of the notional figure. This includes credits for joint implementation and clean development mechanism projects besides domestic actions. Only a very small part of the Kyoto targets is likely to be met through domestic actions in Annex I countries. The Kyoto Protocol, as given final shape in Marrakesh, will at best lead to a marginal reduction in the actual GHG emissions of ratifying Annex I countries.

We report here a preliminary study on the interaction between two common enteric bacteria with the carp Cirrhinus mrigala, a major product of the sewage-fed fish-culture ponds of east Calcutta. The threshold concentration of bacteria in water above which they start appearing in fish muscle was determined. Beyond the threshold value, the concentration of bacteria in muscle varied linearly with the bacterial concentration in the water. A relationship was observed between the concentrations of bacteria in muscle and in the digestive tract with contents.

When recA protein pairs circular single strands with linear duplex DNA, the circular strand displaces its homolog from only one end of the duplex molecule and rapidly creates heteroduplex joints that are thousands of base pairs long [DasGupta, C., Shibata, T., Cunningham, R. P. & Radding, C. M. (1980) Cell 22, 437-446]. To examine this apparently polar reaction, we prepared chimeric duplex fragments of DNA that had M13 nucleotide sequences at one end and G4 sequences at the other. Circular single strands homologous to M13 DNA paired with a chimeric fragment when M13 sequences were located at the 3' end of the complementary strand but did not pair when the M13 sequences were located at the 5' end. Likewise circular single-stranded G4 DNA paired with chimeric fragments only when G4 sequences were located at the 3' end of the complementary strand. To confirm these observations, we prepared fd DNA labeled only at the 5' or 3' end of the plus strand, and we examined the susceptibility of these labeled ends to digestion by exonucleases when joint molecules were formed. Eighty percent of the 5' label in joint molecules became sensitive to exonuclease VII. Displacement of that 5' end by recA protein was concerted because it did not occur in the absence of single-stranded DNA or in the presence of heterologous single strands. By contrast, only a small fraction of the 3' label became sensitive to exonuclease VII or exonuclease I. These observations show that recA protein forms heteroduplex joints in a concerted and polarized way.

The yeast Saccharomyces cerevisiae CDC25 gene encodes a nucleotide-exchange-factor (NEF) that can convert the inactive GDP-bound state of RAS proteins to an active RAS-GTP complex. CDC25 can activate the yeast RAS proteins as well as the human H-ras protein. CDC25 is a member of a family of yeast genes that likely encode NEFs capable of regulating the RAS-related proteins found in yeast. By aligning the amino acid sequence of CDC25-related gene products we found a number of conserved motifs. Using degenerate oligonucleotides that encode these conserved sequences, we have used polymerase chain reactions to amplify fragments of mouse and human cDNAs related to the yeast CDC25 gene. We show that a chimeric molecule, part mouse and part yeast CDC25, can suppress the loss of CDC25 function in the yeast S. cerevisiae.

Escherichia coli recA protein makes joint molecules from single-stranded circular phage DNA (viral or plus strand) and homologous linear duplex DNA by a polar reaction that displaces the 5' end of the plus strand from the duplex molecule [Kahn, R., Cunningham, R. P., DasGupta, C. & Radding, C. M. (1981) Proc. Natl. Acad. Sci. USA 78, 4786-4790]. Growth of the heteroduplex joint, which results from strand exchange or branch migration, stopped at the borders of regions of nonhomologous DNA that were variously located 145, 630, or 1202 nucleotides from the end. Accumulation of migrating branches at heterologous borders demonstrates that their migration is not the result of random diffusion but is actively driven by recA protein. Growth of the heteroduplex joint was blocked even when a heterologous insertion was located in the single-stranded DNA, a case in which the flexible single-stranded region might conceivably fold out of the way under some condition. The recA protein did not make joint molecules from phage phi X174 and G4DNAs, which are 70% homologous, but did join phage fd and M13DNAs, which are 97% homologous. In the latter case, heteroduplex joints extended through regions containing isolated mismatched base pairs but stopped in a region where the fd and M13 sequences differ by an average of 1 base pair in 10. These results suggest that in genetic recombination the discrimination of perfect or near-perfect homology from a high degree of relatedness may be attributable in part to the mechanism by which recA protein promotes strand transfer.

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recA protein, which is essential for general genetic recombination in Escherichia coli, promotes the homologous pairing of single-stranded DNA with double-stranded DNA to form a D loop. The amount of recA protein required for the reaction was directly proportional to the amount of single stranded DNA and was unaffected by similar variations in the amount of double-stranded DNA. The ATP analog, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), which was not rapidly hydrolyzed by recA protein, blocked the formation of D loops but promoted the formation of stable complexes of recA protein and single-stranded DNA. These complexes, in turn, bound homologous or heterologous double-stranded DNA and partially unwound it. Because ATP gamma S competitively inhibited the ATPase activity of recA protein (Km/Ki approximately 300), we infer that ATP gamma S binds at a site that overlaps the site for ATP and that the functional complexes formed in the presence of the analog probably represent partial steps in the overall reaction. If the complexes formed in the presence of ATP gamma S reflect natural intermediates in the formation of D loops, recA protein must promote homologous pairing either by moving juxtaposed single-stranded and double-stranded DNA relative to one another or by forming and dissociating complexes reiteratively until a homologous match occurs.

Purified Escherichia coli recA protein catalyzed ATP-dependent pairing of superhelical DNA and homologous single-stranded fragments. The product of the reaction: (i) was retained by nitrocellulose filters in 1.5 M NaCl/0.15 M Na citrate at pH 7, (ii) was dissociated at pH 12.3 but was not dissociated by heating at 55 degrees C for 4 min or by treatment with 0.2% sodium dodecyl sulfate and proteinase K, (iii) contained covalently closed circular double-stranded DNA (form I DNA), (iv) contained single-stranded fragments associated with replicative form (RF) DNA, and (v) contained a significant fraction of D-loops as judged by electron microscopy. Linear and nicked circular double-stranded DNA did not substitute well for superhelical DNA; intact circular single-stranded DNA did not substitute well for single-stranded fragments. Homologous combinations of single-stranded fragments and superhelical DNA from phages phiX174 and fd reacted, whereas heterologous combinations did not. The reaction required high concentrations of protein and MgCl2. The ATPase activity of purified recA protein was more than 98% dependent on the addition of single-stranded DNA. In 1 mM MgCl2, the ability of superhelical DNA to support the ATPase activity was two-thirds as good as that of single-stranded DNA.

Escherichia coli single-strand binding protein (SSB) or phage T4 gene 32 protein reduced the amount of recA protein required to catalyze the formation of D loops from double-stranded DNA and homologous single-stranded fragments. Neither SSB nor gene 32 protein alone catalyzed the formation of D loops, and excessive amounts of either protein, amounts that were sufficient to saturate the single strands, inhibited the formation of D loops completely. Both the stimulatory activity and the inhibitory activity of SSB resisted boiling, which is consistent with the known thermal stability of SSB, whereas the gene 32 protein was inactivated by heating. The formation of D loops in the presence of both recA protein and SSB required homologous DNA and ATP. Spermidine aided the combined action of SSB and recA protein in forming D loops, but Mg2+ alone was sufficient as a counterion.

Escherichia coli ribosomes were used to refold denatured lactate dehydrogenase from porcine muscle. This activity of ribosomes, unlike most of the chaperons, did not require the presence of ATP. The molar concentration of ribosomes required for this refolding was comparable with that of the enzyme. Restoration of the enzyme activity was demonstrated using assays for both the forward and backward reactions. Binding of the denatured enzyme to ribosomes and its refolding were fairly rapid processes as revealed by the time course of the reaction and inhibition of folding when the denatured enzyme was allowed to refold spontaneously for short times before the addition of ribosomes. This protein-folding activity was detected in 70 S ribosomes as well as its RNA, in 50 S particles and in 23 S rRNA. However, 30 S particles failed to refold the enzyme.

In the presence of adenosine 5'-[gamma-thio]triphosphate, a nonhydrolyzable analog of ATP, Escherichia coli recA protein extensively unwinds duplex DNA in a reaction that is strongly stimulated by either homologous or heterologous single-stranded DNA [Cunningham, R.P., Shibata, T., DasGupta, C. & Radding, C.M. (1979) Nature (London) 281, 191-195]. In the presence of ATP and homologous circular single-stranded DNA, recA protein also unwinds circular duplex DNA that is nicked at a heterologous site. When DNA ligase seals this nick, the product is a highly negatively superhelical molecule that can be relaxed by E. coli topoisomerase I. This unwinding requires a high degree of homology since phi X174 single-stranded DNA does not serve as a cofactor in the unwinding of G4 DNA, even though these molecules are 70% homologous. Like synapsis itself, and unlike strand exchange which follows synapsis, unwinding is sensitive to inhibition by ADP. Because recA protein unwinds duplex DNA when neither the single-stranded DNA nor the duplex DNA has a free end in the region of homology, unwinding can be initiated or mediated by a synaptic structure that differs from that of a simple D loop. The paired circular single strand in the synaptic structure behaves like one strand of an under-wound helix because E. coli topoisomerase I can interwind it with its complement.