Femtosecond all-optical synchronization of an X-ray free-electron laser.

Many advanced applications of X-ray free-electron lasers require pulse durations and time resolutions of only a few femtoseconds. To generate these pulses and to apply them in time-resolved experiments, synchronization techniques that can simultaneously lock all independent components, including all accelerator modules and all external optical lasers, to better than the delivered free-electron laser pulse duration, are needed. Here we achieve all-optical synchronization at the soft X-ray free-electron laser FLASH and demonstrate facility-wide timing to better than 30 fs r.m.s. for 90 fs X-ray photon pulses. Crucially, our analysis indicates that the performance of this optical synchronization is limited primarily by the free-electron laser pulse duration, and should naturally scale to the sub-10 femtosecond level with shorter X-ray pulses.

Sequence analysis of the Lactobacillus plantarum bacteriophage PhiJL-1.

The complete genomic sequence of a Lactobacillus plantarum virulent phage PhiJL-1 was determined. The phage possesses a linear, double-stranded, DNA genome consisting of 36,677 bp with a G+C content of 39.36%. A total of 52 possible open reading frames (ORFs) were identified. According to N-terminal amino acid sequencing and bioinformatic analyses, proven or putative functions were assigned to 21 ORFs (41%), including 5 structural protein genes. The PhiJL-1 genome shows functionally related genes clustered together in a genome structure composed of modules for DNA replication, DNA packaging, head and tail morphogenesis, and lysis. This type of modular genomic organization was similar to several other phages infecting lactic acid bacteria. The structural gene maps revealed that the order of the head and tail genes is highly conserved among the genomes of several Siphoviridae phages, allowing the assignment of probable functions to certain uncharacterized ORFs from phage PhiJL-1 and other Siphoviridae phages.

Human chromosome 19 and related regions in mouse: conservative and lineage-specific evolution.

To illuminate the function and evolutionary history of both genomes, we sequenced mouse DNA related to human chromosome 19. Comparative sequence alignments yielded confirmatory evidence for hypothetical genes and identified exons, regulatory elements, and candidate genes that were missed by other predictive methods. Chromosome-wide comparisons revealed a difference between single-copy HSA19 genes, which are overwhelmingly conserved in mouse, and genes residing in tandem familial clusters, which differ extensively in number, coding capacity, and organization between the two species. Finally, we sequenced breakpoints of all 15 evolutionary rearrangements, providing a view of the forces that drive chromosome evolution in mammals.

High-throughput plasmid purification for capillary sequencing.

The need for expeditious and inexpensive methods for high-throughput DNA sequencing has been highlighted by the accelerated pace of genome DNA sequencing over the past year. At the Joint Genome Institute, the throughput in terms of high-quality bases per day has increased over 20-fold during the past 18 mo, reaching an average of 18.3 million bases per day. To support this unprecedented scaleup, we developed an inexpensive automated method for the isolation and purification of double-stranded plasmid DNA clones for sequencing that is tailored to meet the more stringent needs of the newer capillary electrophoresis DNA sequencing machines. The protocol is based on the magnetic bead method of solid phase reversible immobilization that has been automated by using a CRS-based robotic system. The method described here has enabled us to meet our increases in production while reducing labor and materials costs significantly.

Comprehensive genome sequence analysis of a breast cancer amplicon.

Gene amplification occurs in most solid tumors and is associated with poor prognosis. Amplification of 20q13.2 is common to several tumor types including breast cancer. The 1 Mb of sequence spanning the 20q13.2 breast cancer amplicon is one of the most exhaustively studied segments of the human genome. These studies have included amplicon mapping by comparative genomic hybridization (CGH), fluorescent in-situ hybridization (FISH), array-CGH, quantitative microsatellite analysis (QUMA), and functional genomic studies. Together these studies revealed a complex amplicon structure suggesting the presence of at least two driver genes in some tumors. One of these, ZNF217, is capable of immortalizing human mammary epithelial cells (HMEC) when overexpressed. In addition, we now report the sequencing of this region in human and mouse, and on quantitative expression studies in tumors. Amplicon localization now is straightforward and the availability of human and mouse genomic sequence facilitates their functional analysis. However, comprehensive annotation of megabase-scale regions requires integration of vast amounts of information. We present a system for integrative analysis and demonstrate its utility on 1.2 Mb of sequence spanning the 20q13.2 breast cancer amplicon and 865 kb of syntenic murine sequence. We integrate tumor genome copy number measurements with exhaustive genome landscape mapping, showing that amplicon boundaries are associated with maxima in repetitive element density and a region of evolutionary instability. This integration of comprehensive sequence annotation, quantitative expression analysis, and tumor amplicon boundaries provide evidence for an additional driver gene prefoldin 4 (PFDN4), coregulated genes, conserved noncoding regions, and associate repetitive elements with regions of genomic instability at this locus.

The home stretch, a first analysis of the nearly completed genome of Rhodobacter sphaeroides 2.4.1.

Rhodobacter sphaeroides 2.4.1 is an alpha-3 purple nonsulfur eubacterium with an extensive metabolic repertoire. Under anaerobic conditions, it is able to grow by photosynthesis, respiration and fermentation. Photosynthesis may be photoheterotrophic using organic compounds as both a carbon and a reducing source, or photoautotrophic using carbon dioxide as the sole carbon source and hydrogen as the source of reducing power. In addition, R. sphaeroides can grow both chemoheterotrophically and chemoautotrophically. The structural components of this metabolically diverse organism and their modes of integrated regulation are encoded by a genome of approximately 4.5 Mb in size. The genome comprises two chromosomes CI and CII (2.9 and 0.9 Mb, respectively) and five other replicons. Sequencing of the genome has been carried out by two groups, the Joint Genome Institute, which carried out shotgun-sequencing of the entire genome and The University of Texas-Houston Medical School, which carried out a targeted sequencing strategy of CII. Here we describe our current understanding of the genome when data from both of these groups are combined. Previous work had suggested that the two chromosomes are equal partners sharing responsibilities for fundamental cellular processes. This view has been reinforced by our preliminary analysis of the virtually completed genome sequence. We also have some evidence to suggest that two of the plasmids, pRS241a and pRS241b encode chromosomal type functions and their role may be more than that of accessory elements, perhaps representing replicons in a transition state.

An overview of the genome of Nostoc punctiforme, a multicellular, symbiotic cyanobacterium.

Nostoc punctiforme is a filamentous cyanobacterium with extensive phenotypic characteristics and a relatively large genome, approaching 10 Mb. The phenotypic characteristics include a photoautotrophic, diazotrophic mode of growth, but N. punctiforme is also facultatively heterotrophic; its vegetative cells have multiple developmental alternatives, including terminal differentiation into nitrogen-fixing heterocysts and transient differentiation into spore-like akinetes or motile filaments called hormogonia; and N. punctiforme has broad symbiotic competence with fungi and terrestrial plants, including bryophytes, gymnosperms and an angiosperm. The shotgun-sequencing phase of the N. punctiforme strain ATCC 29133 genome has been completed by the Joint Genome Institute. Annotation of an 8.9 Mb database yielded 7432 open reading frames, 45% of which encode proteins with known or probable known function and 29% of which are unique to N. punctiforme. Comparative analysis of the sequence indicates a genome that is highly plastic and in a state of flux, with numerous insertion sequences and multilocus repeats, as well as genes encoding transposases and DNA modification enzymes. The sequence also reveals the presence of genes encoding putative proteins that collectively define almost all characteristics of cyanobacteria as a group. N. punctiforme has an extensive potential to sense and respond to environmental signals as reflected by the presence of more than 400 genes encoding sensor protein kinases, response regulators and other transcriptional factors. The signal transduction systems and any of the large number of unique genes may play essential roles in the cell differentiation and symbiotic interaction properties of N. punctiforme.

Gene expression analysis by transcript profiling coupled to a gene database query.

We describe an mRNA profiling technique for determining differential gene expression that utilizes, but does not require, prior knowledge of gene sequences. This method permits high-throughput reproducible detection of most expressed sequences with a sensitivity of greater than 1 part in 100,000. Gene identification by database query of a restriction endonuclease fingerprint, confirmed by competitive PCR using gene-specific oligonucleotides, facilitates gene discovery by minimizing isolation procedures. This process, called GeneCalling, was validated by analysis of the gene expression profiles of normal and hypertrophic rat hearts following in vivo pressure overload.

Amino-acid substitutions in a surface turn modulate protein stability.

A surface turn position in a four-helix bundle protein, Rop, was selected to investigate the role of turns in protein structure and stability. Although all twenty amino acids can be substituted at this position to generate a correctly folded protein, they produce an unusually large range of thermodynamic stabilities. Moreover, the majority of substitutions give rise to proteins with enhanced thermal stability compared to that of the wild type. By introducing the same twenty mutations at this position, but in a simplified context, we were able to deconvolute intrinsic preferences from local environmental effects. The intrinsic preferences can be explained on the basis of preferred backbone dihedral angles, but local environmental context can significantly modify these effects.

Redesigning the topology of a four-helix-bundle protein: monomeric Rop.

The topology of alpha-helices and beta-sheets in folded proteins is largely specified by the connectivities of the loops and turns which join them. We have used the protein Rop to test the feasibility of using short glycine-rich linkers to reconnect the alpha-helices within a four-helix-bundle protein. In wild-type Rop the four-helix-bundle structure is formed by the association of two identical helix-turn-helix monomers. Our redesigns encode Rop as a single chain to create a monomeric, rather than a dimeric, form of the protein. Characterization of a series of such variants demonstrates that new connections of this type can be used to generate stable, native-like proteins. The length of the connections is of key importance; if the loops are too short, correct association of the helices is prevented, and misfolded, higher order oligomers occur. Designs with sufficiently long loop connections, however, generate exclusively the desired monomeric form of the protein. Moreover, the successful monomeric designs bind Rop's RNA substrate with affinities that are equal to that of the wild-type protein. This result provides strong confirmation that the positioning of the helices in the monomeric variants is closely similar to that in wild-type Rop.

Cooperative interaction of oestrogen receptor 'zinc finger' domain polypeptides on DNA binding.

The consensus oestrogen response element (ERE) contains two inverted copies of an AGGTCA consensus hexameric half-site, spaced by three base pairs. It differs from many other hormone response elements, such as consensus thyroid (TREp) and retinoic acid (DR-5 RARE) response elements, only in the relative spacing and orientation of these sequences. In the present study we report values for cooperativity (omega) of an oestrogen receptor DNA-binding domain polypeptide upon binding to these sequences. The polypeptide binds with negative cooperativity, or without cooperativity to retinoic acid and thyroid response elements respectively, but with high cooperativity to the ERE. We have also examined cooperativity upon binding of the polypeptide to an ERE variant. Since naturally occurring EREs commonly contain one hexamer which is considerably more degenerate than the other, we designed a hybrid response element in which one hexamer is a consensus ERE, while specific mutations were introduced into the other. We chose to mutate the second half-site to a glucocorticoid response element (GRE) half-site sequence (AGAACA), since normally no binding of the DNA-binding domain polypeptide to a GRE hexamer alone can be detected. In the hybrid response element, however, the GRE half-site is recognized with relatively high affinity, although binding to this sequence is dependent on the previous binding of a polypeptide to the ERE hexamer. Thus, cooperative interactions are capable of mediating the recognition of ERE sequence degeneracy. The ability of protein-protein interactions to mediate recognition of DNA sequence degeneracy may also have implications for transcription factors in general.

Dissecting RNA-protein interactions: RNA-RNA recognition by Rop.

The ColE1 plasmid of E. coli encodes a small RNA-binding protein, Rop, which is involved in the regulation of plasmid copy number. Rop, a 4-helix bundle protein, facilitates sense-antisense RNA pairing by binding to the transiently formed hairpin pairs of RNA I and the complementary RNA II. We have identified the residues of Rop that are involved in RNA recognition. The residues form a narrow stripe down one face of the bundle and are symmetrically arranged, with recognition centered about two phenylalanine residues. Our results suggest that these phenylalanine residues interact with the loop region of the hairpin pair, with additional interactions between eight polar residues and the phosphate backbone. By modifying the identity of residue 14, we have created a variant of Rop that displays altered RNA binding specificity. The results of our studies allow us to present a detailed picture of RNA-protein recognition in a novel model system.

Metal replacement in "zinc finger" and its effect on DNA binding.

Metal replacement studies were used to investigate the metal requirement of a bacterially expressed polypeptide encoding the zinc finger DNA binding domain of the estrogen receptor. Apopolypeptide was generated by dialysis of native polypeptide against low-pH buffer under reducing conditions. Specific DNA binding can be restored by refolding the apopolypeptide in the presence of ionic zinc, cadmium, or cobalt. However, refolding in the presence of copper or nickel fails to regenerate DNA binding activity. While cobalt-reconstituted polypeptide has a reduced affinity for its AGGTCA-binding site compared to zinc- or cadmium-polypeptide, it has the surprising property of increased cooperative DNA binding. Our work indicates that metal substitution results in a range of effects upon DNA binding in vitro. The potential biological significance of metal substitution in vivo is discussed.

ColE1-compatible vectors for high-level expression of cloned DNAs from the T7 promoter.

A new family of T7-based expression plasmids with unique features is described. The plasmid origin of replication (ori), derived from P15A, is compatible with that of ColE1-derived plasmids, which facilitates the co-production of proteins from these vectors and from ColE1-derived T7 expression vectors in the same cell. The plasmids are medium-copy-number and also carry the M13 ori. Consequently, both double- and single-stranded DNA can be easily obtained. The plasmids encode KmR, thus avoiding the potential for plasmid loss associated with ApR-based systems. One of the plasmids carries the lacI gene, to allow for more stringent regulation of the production of potentially toxic proteins. When the plasmids are introduced into an Escherichia coli strain such as BL21(DE3), which contains the T7 polymerase-encoding gene under control of the lacUV5 promoter, addition of IPTG initiates the production of high levels of the recombinant protein.

Ordered binding of retinoic acid and retinoid-X receptors to asymmetric response elements involves determinants adjacent to the DNA-binding domain.

Retinoic acid, a pleiotropic regulator of development and homeostasis, controls the expression of specific gene networks via direct interactions with nuclear receptors. The retinoic acid receptor (RAR), as a heterodimer with the retinoid-x receptor (RXR), binds to DNA recognition sites, referred to as retinoic acid response elements (RAREs), that are generally composed of a direct repeat of the half-site core motif PuGGTCA spaced by 2 (DR-2) or 5 (DR-5) basepairs. The asymmetric nature of direct repeat RAREs suggests that RAR and RXR bind preferentially to one of the two half-site core motifs. Here we show that RXR occupies the 5'-up-stream half-site, and RAR the 3'-down-stream half-site of the direct repeat in both DR-2 and DR-5 RAREs. We also demonstrate that a region adjacent to the zinc finger region of RAR and RXR is essential for specific and cooperative binding of DNA-binding domain peptides to RAREs. However, differential utilization of these determinants mediate RAR-RXR heterodimer binding to DR-2 and DR-5 RAREs. The demonstration of ordered but nonequivalent binding of RAR-RXR complexes to DR-2 and DR-5 RAREs sets a precedent for the generation of sequence specificities in heterodimeric DNA-binding proteins.

Further characterization of the N-terminal copper(II)- and nickel(II)-binding motif of proteins. Studies of metal binding to chicken serum albumin and the native sequence peptide.

We have investigated the Cu(II)- and Ni(II)-binding properties of chicken serum albumin (CSA) and of the native sequence tripeptide derived from the N-terminus of this protein. Spectrophotometric and equilibrium dialysis experiments demonstrate that Cu(II) and Ni(II) bind non-specifically at the N-terminus of CSA. Proton displacement studies show that the histidine residue in the fourth position of the protein does not appear to participate in the binding of the two metals. Consistent results were obtained with the native sequence tripeptide L-aspartyl-L-alanyl-L-glutamic acid N-methylamide. The results presented here demonstrate that neither the glutamic acid residue in the third position nor the histidine in the fourth position participate in the binding of Cu(II) and Ni(II) to CSA. It is known, however, that a number of other albumins with a histidine residue in the third position possess high-affinity Cu(II)- and Ni(II)-binding sites. Our results provide further evidence that the N-terminal Cu(II)/Ni(II)-binding motif requires a histidine at the third position in order to bind Cu(II) and Ni(II) specifically.

Effect of replacement of "zinc finger" zinc on estrogen receptor DNA interactions.

Exposure of bovine estrogen receptor to the metal chelators EDTA and 1,10-phenanthroline results in a loss of nonspecific DNA binding, presumably because of the removal of "zinc finger" zinc. Nonspecific DNA binding, as measured by a DNA-cellulose binding assay, can be restored by dialysis of the aporeceptor against buffer containing zinc, cadmium, and cobalt but not with buffer containing copper or nickel. More detailed studies were carried out using a bacterially expressed polypeptide encompassing the DNA binding domain of the human estrogen receptor. Apopolypeptide fails to bind DNA specifically, as measured by mobility shift assay using a consensus estrogen response element hexamer containing oligonucleotide, but DNA binding was restored by dialysis of the apopolypeptide against buffer containing zinc, cadmium, and cobalt but not with buffer containing copper or nickel. Dissociation constants of zinc- and cadmium-reconstituted polypeptide for the estrogen response element hexamer (66 and 48 nM, respectively) are virtually indistinguishable from native polypeptide (Kd = 48 nM) whereas cobalt-reconstituted polypeptide has a lower affinity (Kd = 720 nM). However, native, zinc-, cadmium-, and cobalt-reconstituted polypeptides gave identical results in a methylation interference assay. Competition experiments with zinc and copper or nickel suggest that copper and nickel are able to bind to zinc finger residues but do so nonproductively. The relative affinities copper greater than cadmium greater than zinc greater than cobalt greater than nickel for the polypeptide were determined by a zinc blot competition assay. The ability of cadmium and cobalt to substitute for zinc in the zinc fingers demonstrates a structural "flexibility" in the DNA binding domain as each of these metals has slightly different ionic radii. On the other hand, subtle differences in DNA binding affinity and/or specificity could exist, which may not be detectable here. Also, the ability of metals to substitute for zinc in the DNA binding domain suggests that metal substitution in these zinc fingers in vivo may be of relevance to the toxicity and/or carcinogenicity of some of these metals.

Characterization and cellular distribution of acidic peptide and oligosaccharide metal-binding compounds from kidneys.

Two low-molecular-mass Ni-binding fractions first isolated from human kidneys [Templeton & Sarkar (1985) Biochem. J. 230, 35-42] are further characterized. Both components are acidic and are readily separated from each other by gel chromatography on Bio-Gel P-2. After equilibration with 63Ni the largest complex constitutes about 30% of the radioactive 63Ni and is an approx. 3.5 kDa peptide and the smallest species comprise short oligosaccharides containing 70% of the radioactivity. Both of these components are found in human, bovine and porcine kidneys as well as in a porcine proximal tubule-like cell line LLC-PK1. There is a small variation in amino acid composition between species. The oligosaccharides are reducing sugars and contain sulphate, glucosamine, glucuronic acid and iduronic acid with two to four overall negative charges. The monosaccharide composition was determined by h.p.l.c. with pulsed amperometric detection of the acid hydrolysates and by gas chromatography. In the LLC-PK1 cell line the acidic peptide is both intracellular and extracellular, whereas the oligosaccharides are only intracellular. The concentration of extracellular peptide, as measured by 63Ni binding, is found to increase after exposure of the cells to low micromolar concentrations of Ni, whereas the oligosaccharide concentrations, also measured by 63Ni binding, remain constant. The oligosaccharide component is decreased by 40% in the presence of NH4Cl, suggesting that is derived from degradation of internalized heparan sulphate.