Bioavailability of water-polluting sulfonoaromatic compounds

Highly substituted arenesulfonates are chemically stable compounds with a range of industrial applications, and they are widely regarded as being poorly degradable. We did enrichment cultures for bacteria able to utilise the sulfonate moiety of 14 compounds, and we obtained mixed cultures that were able to desulfonate each compound. The products formed were usually identified as the corresponding phenol, but because we could not obtain pure cultures, we followed up these findings with quantitative work in pure cultures of, e.g., Pseudomonas putida S-313, which generated the same phenols from the compounds studied. Many of these phenols are known to be biodegradable, or to be subject to binding to soil components. We thus presume that the capacity to degrade aromatic sulfonates extensively is widespread in the environment, even though the degradative capacity is spread over several organisms and conditions. Received: 9 February 1999 / Revision received: 7 April 1999 / Accepted: 9 April 1999     

The two zinc fingers in the human immunodeficiency virus type 1 nucleocapsid protein are not functionally equivalent.

The highly conserved zinc fingers in retroviral nucleocapsid (NC) proteins have the general structure Cys-(X)2-Cys-(X)4-His-(X)4-Cys. Human immunodeficiency virus type 1 (HIV-1) contains two Zn2+ fingers, and mutants were constructed in which the native sequence of each Zn2+ finger was maintained but their positions in the NC protein were changed. Mutants had either two first-finger sequences (pNC1/1), two second-finger sequences (pNC2/2), or reversed first- and second-finger sequences (pNC2/1). Cells transfected with mutant or wild-type clones produced similar levels of Tat, Gag, Pol, and Env proteins, formed syncytia, and shed viruslike particles that were indistinguishable by electron microscopy. However, the pNC2/1 and pNC2/2 mutants were inefficient in packaging genomic RNA (less than 15% of wild-type levels), whereas the pNC1/1 mutant packaged approximately 70% of wild-type levels of RNA. No infectious virus could be detected with either the pNC2/1 or pNC2/2 mutants, whereas the pNC1/1 mutant appeared to sustain a low level of replication and reverted to a competent wild-type-like viral species after a 2- to 4-week lag period. The data strongly suggest that the two Zn2+ fingers of HIV-1 are not functionally equivalent and that the first Zn2+ finger in the Gag precursor plays a more prominent role in RNA selection and packaging. The data also indicate that both Zn2+ fingers in the mature NC protein play as yet unknown roles in viral assembly or the early stages of the viral infection process.     

Nuclear factor I (NF I) binds to an NF I-type site but not to the CCAAT site in the human alpha-globin gene promoter.

Methylation interference and missing contact analyses demonstrate that nuclear factor I (NF I) recognizes an NF I-like site (5'-GGG(N)6GCCAG-3') within the alpha-globin promoter rather than the adjacent CCAAT box. Consistent with this, mutations within the CCAAT box do not alter significantly the affinity and specificity of the interaction whereas elimination of the 5'-GGG-3' half-site of the recognition sequence reduces the DNA binding strength of NF I by 2 orders of magnitude down to the range of unspecific interaction. On the other hand, the mutated alpha-globin promoter sequence that is no longer bound by NF I, although it retains an intact CCAAT box, interacts specifically with a protein component from nuclear extracts of HeLa cells. From these results we conclude that NF I is not the factor that interacts with the CCAAT box and that the second half of the canonical 5'-TGG(N)6GCCAA-3' NF I binding site cannot be regarded as identical with the CCAAT promoter element, as suggested previously.     

Basis for receptor specificity of nonecotropic murine leukemia virus surface glycoprotein gp70SU.

Murine leukemia viruses (MuLVs) initiate infection of NIH 3T3 cells by binding of the viral envelope (Env) protein to a cell surface receptor. Interference assays have shown that MuLVs can be divided into four groups, each using a distinct receptor: ecotropic, polytropic, amphotropic, and 10A1. In this study, we have attempted to map the determinants within viral Env proteins by constructing chimeric env genes. Chimeras were made in all six pairwise combinations between Moloney MCF (a polytropic MuLV), amphotropic MuLV, and 10A1, using a conserved EcoRI site in the middle of the Env coding region. The receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity of each chimera was determined by using an interference assay. We found that amphotropic receptor specificity seems to map to the N-terminal portion of surface glycoprotein gp70SU. The difference between amphotropic and 10A1 receptor specificity can be attributed to one or more of only six amino acid differences in this region. Nearly all other cases showed evidence of interaction between Env domains in the generation of receptor specificity. Thus, a chimera composed exclusively of MCF and amphotropic sequences was found to exhibit 10A1 receptor specificity. None of the chimeras were able to infect cells by using the MCF receptor; however, two chimeras containing the C-terminal portion of MCF gp70SU could bind to this receptor, while they were able to infect cells via the amphotropic receptor. This result raises the possibility that receptor binding maps to the C-terminal portion of MCF gp70SU but requires MCF N-terminal sequences for a functional interaction with the MCF receptor.     

Regulatory control and the costs and benefits of biochemical noise

Experiments in recent years have vividly demonstrated that gene expression can be highly stochastic. How protein concentration fluctuations affect the growth rate of a population of cells is, however, a wide-open question. We present a mathematical model that makes it possible to quantify the effect of protein concentration fluctuations on the growth rate of a population of genetically identical cells. The model predicts that the population's growth rate depends on how the growth rate of a single cell varies with protein concentration, the variance of the protein concentration fluctuations, and the correlation time of these fluctuations. The model also predicts that when the average concentration of a protein is close to the value that maximizes the growth rate, fluctuations in its concentration always reduce the growth rate. However, when the average protein concentration deviates sufficiently from the optimal level, fluctuations can enhance the growth rate of the population, even when the growth rate of a cell depends linearly on the protein concentration. The model also shows that the ensemble or population average of a quantity, such as the average protein expression level or its variance, is in general not equal to its time average as obtained from tracing a single cell and its descendants. We apply our model to perform a cost-benefit analysis of gene regulatory control. Our analysis predicts that the optimal expression level of a gene regulatory protein is determined by the trade-off between the cost of synthesizing the regulatory protein and the benefit of minimizing the fluctuations in the expression of its target gene. We discuss possible experiments that could test our predictions.     

HIV-1 Gag extension: conformational changes require simultaneous interaction with membrane and nucleic acid

The retroviral Gag polyprotein mediates viral assembly. The Gag protein has been shown to interact with other Gag proteins, with the viral RNA, and with the cell membrane during the assembly process. Intrinsically disordered regions linking ordered domains make characterization of the protein structure difficult. Through small-angle scattering and molecular modeling, we have previously shown that monomeric human immunodeficiency virus type 1 (HIV-1) Gag protein in solution adopts compact conformations. However, cryo-electron microscopic analysis of immature virions shows that in these particles, HIV-1 Gag protein molecules are rod shaped. These differing results imply that large changes in Gag conformation are possible and may be required for viral formation. By recapitulating key interactions in the assembly process and characterizing the Gag protein using neutron scattering, we have identified interactions capable of reversibly extending the Gag protein. In addition, we demonstrate advanced applications of neutron reflectivity in resolving Gag conformations on a membrane. Several kinds of evidence show that basic residues found on the distal N- and C-terminal domains enable both ends of Gag to bind to either membranes or nucleic acid. These results, together with other published observations, suggest that simultaneous interactions of an HIV-1 Gag molecule with all three components (protein, nucleic acid, and membrane) are required for full extension of the protein.     

The CW domain, a new histone recognition module in chromatin proteins

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.     

On the Role of the SP1 Domain in HIV-1 Particle Assembly: a Molecular Switch?

Expression of a retroviral protein, Gag, in mammalian cells is sufficient for assembly of immature virus-like particles (VLPs). VLP assembly is mediated largely by interactions between the capsid (CA) domains of Gag molecules but is facilitated by binding of the nucleocapsid (NC) domain to nucleic acid. We have investigated the role of SP1, a spacer between CA and NC in HIV-1 Gag, in VLP assembly. Mutational analysis showed that even subtle changes in the first 4 residues of SP1 destroy the ability of Gag to assemble correctly, frequently leading to formation of tubes or other misassembled structures rather than proper VLPs. We also studied the conformation of the CA-SP1 junction region in solution, using both molecular dynamics simulations and circular dichroism. Consonant with nuclear magnetic resonance (NMR) studies from other laboratories, we found that SP1 is nearly unstructured in aqueous solution but undergoes a concerted change to an α-helical conformation when the polarity of the environment is reduced by addition of dimethyl sulfoxide (DMSO), trifluoroethanol, or ethanol. Remarkably, such a coil-to-helix transition is also recapitulated in an aqueous medium at high peptide concentrations. The exquisite sensitivity of SP1 to mutational changes and its ability to undergo a concentration-dependent structural transition raise the possibility that SP1 could act as a molecular switch to prime HIV-1 Gag for VLP assembly. We suggest that changes in the local environment of SP1 when Gag oligomerizes on nucleic acid might trigger this switch.