Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.     

Location and analysis of nucleotide sequences at one end of a putative lac transposon in the Escherichia coli chromosome.

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon Tn5 and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a site-specific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.     

Gene organization and structure of the Streptomyces lividans gal operon.

We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism.     

Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes.

The fixA, fixB, fixC, and fixX genes of Rhizobium meliloti 1021 constitute an operon and are required for nitrogen fixation in alfalfa nodules. DNA homologous to the R. meliloti fixABC genes is present in all other Rhizobium and Bradyrhizobium species examined, but fixABC-homologous sequences were found in only one free-living diazotroph, Azotobacter vinelandii. To determine whether the fixABCX genes share sequence homology with any of the 17 Klebsiella pneumoniae nif genes, we determined the entire nucleotide sequence of the fixA, fixB, fixC, and fixX genes and defined four open reading frames that code for polypeptides of molecular weights 31,146, 37,786, 47,288, and 10,937, respectively. Neither DNA nor amino acid sequence homology to the R. meliloti fixA, -B, -C, and -X genes was found in the K. pneumoniae nif operon. The fixX gene contains a cluster of cysteine residues characteristic of ferredoxins and is highly homologous to an Azotobacter ferredoxin which has been shown to donate electrons to nitrogenase. The fixABC operon contains a promoter region that is highly homologous to other nifA-activated promoters. We also found a duplication of the 5' end of the fixABCX operon; a 250-bp region located 520 bp upstream of the fixABCX promoter bears more than 65% homology to the 5' end of the transcribed region, including the first 32 codons of fixA.     

IS1 insertion generates duplication of a nine base pair sequence at its target site.

Three independent integrations of the E. coli insertion sequence, IS1, into the gal operon have been analyzed. DNA sequences of portions of the wild-type galT gene which act as the target sites for these insertions, as well as the corresponding gal/IS1 junctions, are reported. Two features are particularly noteworthy. First, similar sequences appearing in inverted orientation consitute the ends of IS1: 18 of the terminal 23 base pairs at each end are identical. Second, in all three insertions, a 9 base pair segment found once in the wild-type sequence at the site of insertion is duplicated and appears in the same orientation at each end of the inserted element. The sequence of this 9 base pair repeat is different for each insertion analyzed. No homology between the inverted repeat sequences at the ends of IS1 and the sequences of the target sites is observed. Models for the mechanism of IS1 insertion are proposed.     

The effect of intrinsic curvature on conformational properties of circular DNA.

Both thermal fluctuations and the intrinsic curvature of DNA contribute to conformations of the DNA axis. We looked for a way to estimate the relative contributions of these two components of the double-helix curvature for DNA with a typical sequence. We developed a model and Monte Carlo procedure to simulate the Boltzmann distribution of DNA conformations with a specific intrinsic curvature. Two steps were used to construct the equilibrium conformation of the model chain. We first specified the equilibrium DNA conformation at the base pair level of resolution, using a set of the equilibrium dinucleotide angles and DNA sequence. This conformation was then approximated by the conformation of the model chain consisting of a reduced number of longer, straight cylindrical segments. Each segment of the chain corresponded to a certain number of DNA base pairs. We simulated conformational properties of nicked circular DNA for different sets of equilibrium dinucleotide angles, different random DNA sequences, and lengths. Only random sequences of DNA generated with equal probability of appearance for all types of bases at any site of the sequence were used. The results showed that for a broad range of intrinsic curvature parameters, the radius of gyration of DNA circles should be nearly independent of DNA sequence for all DNA lengths studied. We found, however, a DNA properly that should strongly depend on DNA sequence if the double helix has essential intrinsic curvature. This property is the equilibrium distribution of the linking number for DNA circles that are 300-1000 bp in length. We found that a large fraction of the distributions corresponding to random DNA sequences should have two separate maxima. The physical nature of this unexpected effect is discussed. This finding opens new opportunities for joined experimental and theoretical studies of DNA intrinsic curvature.     

A new moderately repetitive DNA sequence family of novel organization.

In cloning adenovirus homologous sequences, from a human cosmid library, we identified a moderately repetitive DNA sequence family consisting of tandem arrays of 2.5 kb members. A member was sequenced and several non-adjacent, 15-20 bp G-C rich segments with homology to the left side of adenovirus were discovered. The copy number of 400 members is highly conserved among humans. Southern blots of partial digests of human DNA have verified the tandem array of the sequence family. The chromosomal location was defined by somatic cell genetics and in situ hybridization. Tandem arrays are found only on chromosomes 4 (4q31) and 19 (q13.1-q13.5). Homologous repetitive sequences are found in DNA of other primates but not in cat or mouse. Thus we have identified a new family of moderately repetitive DNA sequences, unique because of its organization in clustered tandem arrays, its length, its chromosomal location, and its lack of homology to other moderately repetitive sequence families.     

Frequency among Enterobacteriaceae of the DNA sequences encoding type 1 pili.

Type 1 pili, characterized by mannose-inhibitable agglutination of fowl or guinea pig erythrocytes, have been found throughout the family Enterobacteriaceae. A radiolabeled probe was prepared from a restriction endonuclease-digested fragment of the Escherichia coli pil operon and used to detect homologous DNA sequences in 236 bacteria representing 11 genera of Enterobacteriaceae. Only isolates identified as E. coli or Shigella spp. exhibited homology. In contrast, mannose-sensitive hemagglutination was observed in nine genera. Probe DNA did not hybridize to plasmid DNA, indicating a chromosomal location for the pil operon. Analysis of restriction nuclease-digested whole-cell DNA from 60 E. coli and two Shigella sp. isolates indicated that internal sequences were conserved in most strains, but that changes in flanking sequences in the chromosome were common.     

High A + T content conserved in DNA sequences upstream of leuABCD in Escherichia coli and Salmonella typhimurium.

The nucleotide sequence of over 800 base pairs of DNA upstream of leuP was determined for Escherichia coli and Salmonella typhimurium. In both of these enteric bacteria, approximately 500 base pairs of A + T-rich sequences separates leuP from an upstream open reading frame. Although these A + T-rich sequences share little homology, the distribution of A + T base pairs within the region is strikingly conserved. Deletion of the A + T-rich sequences upstream of the E. coli leu operon does not markedly affect the strength of the leu promoter in vivo.     

Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in Gram-positive bacteria

A 1.5 kb cryptic plasmid was isolated from Helicobacter pylori. Low-stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication-initiation proteins commonly found in small plasmids endogenous to Gram-positive bacteria which replicate by the 'rolling-circle' mechanism. Sequence motifs corresponding to the origins-of-replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram-negative bacterium to show replication determinants characteristic of the 'rolling-circle' group of plasmids from Gram-positive bacteria. The cloned plasmid will be used to develop a shuttle-vector for H. pylori.     

DNA sequences at the sites of three insertions of the transposable element Tn5 in the histidine operon of Salmonella

Summary The DNA sequence has been determined at the sites of three independent occurrences of the transposable element Tn5 in the hisG gene of the histidine operon. All three insertion sites are adjacent to G+C-rich stretches of DNA. In all three cases sequences at the sites of insertion show homology with a region near the ends of Tn5.     

Sequence and temperature effect on hydrogen bond disruption in DNA determined by a statistical analysis

We use the modified self-consistent phonon approximation theory to calculate temperature dependent interbase hydrogen bond disruption profiles for a number of six base pair repeating sequence infinite B-DNA polymers with various guanine-cytosine/adenine-thymine ratios. For comparison we also include results we have obtained in our earlier work on several B-DNA homopolymers, copolymers and a four-base-pair repeating sequence polymer. Our theory gives a statistical estimate of thermal fluctuational disruption probability of individual hydrogen bonds in individual base pairs in DNA as a function of temperature. The calculated probabilities show no sequence dependence at premelting temperatures, in agreement with proton exchange measurements. These probabilities however become very sensitive to base sequence at temperatures close to the observed melting temperatures. Multi-phasic critical transitions are found in which a portion of base pairs are disrupted at temperatures below the final disruption temperature. These transitions include localized as well as non-localized base pair opening. The localized transitions involve disruption of a few base-pairs at every other location without large scale base unstacking, and they may not appear in the observed UV curves with current resolution. On the other hand the overall disruption behavior is consistent with observations. The midpoint transition temperatures are close to the observed melting temperatures and these temperatures show the observed linear dependence on guanine-cytosine content. Our calculations indicate that our theory can be used effectively to calculate H-bond disruption behavior of different DNA sequences. Received: 20 February 1996 / Accepted: 2 May 1996     

Distribution of sequences homologous to the impCAB operon of TP110 among bacterial plasmids of different incompatibility groups

Summary Mutagenic DNA repair is a function of many naturally occurring plasmids belonging to several different incompatibility groups. A DNA probe corresponding to the impCAB operon of the IncIl plasmid TP110, which encodes such functions, was used to investigate the distribution of homologous sequences in both related and unrelated plasmids. Southern blotting was used to demonstrate considerable sequence conservation amongst a number of plasmid types, with imp-related sequences being found on plasmids belonging to the I1, I1/B, B and FIV incompatibility groups. However, no homology was detected amongst plasmids of the N and L/M incompatibility groups, many of which carry functionally similar gene clusters. It appears that sequences determining mutagenic repair functions have been largely conserved within any one incompatibility group, but that significant divergent evolution has occurred between groups.     

A new rice repetitive DNA shows sequence homology to both 5S RNA and tRNA.

Moderately repetitive DNA sequences are found in the genomes of all eucaryotes that have been examined. We now report the discovery of a novel, transcribed, moderately repetitive DNA sequence in a higher plant which is different from any of the known repetitive DNA sequences from any organism. We isolated a rice cDNA clone which hybridizes to multiple bands on genomic blot analysis. The sequence of this 352 bp cDNA contains four regions of homology to the wheat phenylalanine tRNA, including the polymerase III-type promoter. Unexpectedly, two regions of the same 352 bp sequence also show homology to the wheat 5S RNA sequence. Using the cDNA as a probe, we have isolated six genomic clones which contain long tandem repeats of 355 bp sequence, and have sequenced nine repeat units. Our findings suggest that the rice repetitive sequence may be an amplified pseudogene with sequence homology to both 5S RNA and tRNA, but organized as long tandem repeats resembling 5S RNA genes. This is the first example showing homology between the sequences of a moderately repetitive DNA with unknown function and 5S RNA.