PubMed Health⌕ Search

Biomedical subjects

K E Rudd

Publications and source records attributed to K E Rudd.

At least 19 recordsLinked to original sources
PubMed2001

Anatomy of Escherichia coli ribosome binding sites.

During translational initiation in prokaryotes, the 3' end of the 16S rRNA binds to a region just upstream of the initiation codon. The relationship between this Shine-Dalgarno (SD) region and the binding of ribosomes to translation start-points has been well studied, but a unified mathematical connection between the SD, the initiation codon and the spacing between them has been lacking. Using information theory, we constructed a model that treats these three components uniformly by assigning to the SD and the initiation region (IR) conservations in bits of information, and by assigning to the spacing an uncertainty, also in bits. To build the model, we first aligned the SD region by maximizing the information content there. The ease of this process confirmed the existence of the SD pattern within a set of 4122 reviewed and revised Escherichia coli gene starts. This large data set allowed us to show graphically, by sequence logos, that the spacing between the SD and the initiation region affects both the SD site conservation and its pattern. We used the aligned SD, the spacing, and the initiation region to model ribosome binding and to identify gene starts that do not conform to the ribosome binding site model. A total of 569 experimentally proven starts are more conserved (have higher information content) than the full set of revised starts, which probably reflects an experimental bias against the detection of gene products that have inefficient ribosome binding sites. Models were refined cyclically by removing non-conforming weak sites. After this procedure, models derived from either the original or the revised gene start annotation were similar. Therefore, this information theory-based technique provides a method for easily constructing biologically sensible ribosome binding site models. Such models should be useful for refining gene-start predictions of any sequenced bacterial genome.

Base Sequence↗
PubMed2000

EcoGene: a genome sequence database for Escherichia coli K-12.

The EcoGene database provides a set of gene and protein sequences derived from the genome sequence of Escherichia coli K-12. EcoGene is a source of re-annotated sequences for the SWISS-PROT and Colibri databases. EcoGene is used for genetic and physical map compilations in collaboration with the Coli Genetic Stock Center. The EcoGene12 release includes 4293 genes. EcoGene12 differs from the GenBank annotation of the complete genome sequence in several ways, including (i) the revision of 706 predicted or confirmed gene start sites, (ii) the correction or hypothetical reconstruction of 61 frame-shifts caused by either sequence error or mutation, (iii) the reconstruction of 14 protein sequences interrupted by the insertion of IS elements, and (iv) pre-dictions that 92 genes are partially deleted gene fragments. A literature survey identified 717 proteins whose N-terminal amino acids have been verified by sequencing. 12 446 cross-references to 6835 literature citations and s are provided. EcoGene is accessible at a new website: http://bmb.med.miami.edu/EcoGene/EcoWeb. Users can search and retrieve individual EcoGene GenePages or they can download large datasets for incorporation into database management systems, facilitating various genome-scale computational and functional analyses.

Databases, Factual↗
PubMed2000

Revised translation start site for secM defines an atypical signal peptide that regulates Escherichia coli secA expression.

The secretion-responsive regulation of Escherichia coli secA occurs by coupling its translation to the translation and secretion of an upstream regulator, secM (formerly geneX). We revise the translational start site for secM, defining a new signal peptide sequence with an extended amino-terminal region. Mutational studies indicate that certain atypical amino acyl residues within this extended region are critical for proper secA regulation.

Adenosine Triphosphatases↗
PubMed1998

The vacB gene required for virulence in Shigella flexneri and Escherichia coli encodes the exoribonuclease RNase R.

vacB, a gene previously shown to be required for expression of virulence in Shigella and enteroinvasive Escherichia coli, has been found to encode the 3'-5' exoribonuclease, RNase R. Thus, cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable. These data indicate that RNase R participates in an essential cell function in addition to its role in virulence. The identification of the vacB gene product as RNase R should aid in understanding how the virulence phenotype in enterobacteria is expressed and regulated. On the basis of this information we propose that vacB be renamed rnr.

Bacterial Proteins↗
PubMed1998

Low molecular weight proteins: a challenge for post-genomic research.

The EcoGene project involves the examination of Escherichia coli K-12 DNA sequences and accompanying annotation in the public databases in order to refine the representation and prediction of the entire set of E. coli K-12 chromosomally encoded protein sequences. The results of this ongoing effort have been deposited in the SWISSPROT protein sequence database as sequencing of the E. coli genome has progressed to completion in recent years. Through this continuing research, we have discovered that the prediction of low molecular weight (small) proteins, arbitrarily defined as protein sequences < or = 150 amino acids (aa) in length, is problematic and requires special attention. We describe the small protein subset of EcoGene and the approach used to derive this subset from the complete E. coli genome sequence and database annotations. These E. coli proteins have helped to identify new small genes in other organisms and to identify conserved residues (motifs) using database searches and multiple alignments. Two thirds of the E. coli small proteins have not been characterized experimentally. The careful application of computer and laboratory methods to the analysis of small proteins is needed for accurate prediction, verification and characterization. The problem of accurate protein sequence identification is not limited to small proteins or to E. coli; these problems are encountered to varying degrees throughout all sequence databases.

Amino Acid Sequence↗
PubMed1998

Linkage map of Escherichia coli K-12, edition 10: the physical map.

A physical map, EcoMap10, of the now completely sequenced Escherichia coli chromosome is presented. Calculated genomic positions for the eight restriction enzymes BamHI, HindIII, EcoRI, EcoRV, BglI, KpnI, PstI, and PvuII are depicted. Both sequenced and unsequenced Kohara/Isono miniset clones are aligned to this calculated restriction map. DNA sequence searches identify the precise locations of insertion sequence elements and repetitive extragenic palindrome clusters. EcoGene10, a revised set of genes and functionally uncharacterized open reading frames (ORFs), is also depicted on EcoMap10. The complete set of unnamed ORFs in EcoGene10 are assigned provisional names beginning with the letter "y" by using a systematic nomenclature.

Chromosome Mapping↗
PubMed1997

A signal transducer for aerotaxis in Escherichia coli.

The newly discovered aer locus of Escherichia coli encodes a 506-residue protein with an N terminus that resembles the NifL aerosensor and a C terminus that resembles the flagellar signaling domain of methyl-accepting chemoreceptors. Deletion mutants lacking a functional Aer protein failed to congregate around air bubbles or follow oxygen gradients in soft agar plates. Membranes with overexpressed Aer protein also contained high levels of noncovalently associated flavin adenine dinucleotide (FAD). We propose that Aer is a flavoprotein that mediates positive aerotactic responses in E. coli. Aer may use its FAD prosthetic group as a cellular redox sensor to monitor environmental oxygen levels.

Bacterial Proteins↗
PubMed1997

The aquaporin-Z water channel gene of Escherichia coli: structure, organization and phylogeny.

Aquaporin water channel proteins are found throughout the plant and animal kingdoms, but the first prokaryotic water channel gene, aqpZ, was only recently identified in wild type Escherichia coli (Calamita G et al (1995) J Biol Chem 270, 29063-29066). Here we define the organization of aqpZ in E coli, produce the AqpZ protein and compare the AqpZ phylogeny to that of some known bacterial homologs. Physical mapping and sequence analyses confirmed the location of aqpZ at minute 19.7 on the E coli chromosome where it is transcribed counterclockwise. The monocistronic nature of aqpZ was clearly indicated by the structural organization of its surrounding genes, ybjD and ybjE' and by the presence of a typical Rho-independent transcriptional terminator following the aqpZ stop codon. Computer sequence analysis indicated the -35/-10 region located 72 bases upstream of the aqpZ start codon as the most likely aqpZ promoter. A series of potential cis-regulatory elements were found in the 400 bp region preceding the aqpZ ORF. The AqpZ protein, produced under T7 phi 10 control, showed a size of about 20 kDa by SDS-PAGE. Striking similarities were found between the E coli aqpZ and a gene included in the genome of the cyanobacterium Synechocystis sp PCC6803, a species permanently living a fresh water. These results may represent a fundamental step to characterize the regulation and the physiological features of the AqpZ water channel in prokaryotes.

Aquaporins↗
PubMed1996

Sequencing and analysis of bacterial genomes.

The complete sequences of two small bacterial genomes have recently become available, and those of several more species should follow within the next two years. Sequence comparisons show that the most bacterial proteins are highly conserved in evolution, allowing predictions to be made about the functions of most products of an uncharacterized genome. Bacterial genomes differ vastly in their gene repertoires. Although genes for components of the translation and transcription machinery, and for molecular chaperones, are typically maintained, many regulatory and metabolic systems are absent in bacteria with small genomes. Mycoplasma genitalium, with the smallest known genome of any cellular life form, lacks virtually all known regulatory genes, and its gene expression may be regulated differently than in other bacteria. Genome organization is evolutionarily labile: extensive gene shuffling leaves only very few conserved gene arrays in distantly related bacteria.

Amino Acid Sequence↗
PubMed1996

Metabolism and evolution of Haemophilus influenzae deduced from a whole-genome comparison with Escherichia coli.

BACKGROUND: The 1.83 Megabase (Mb) sequence of the Haemophilus influenzae chromosome, the first completed genome sequence of a cellular life form, has been recently reported. Approximately 75 % of the 4.7 Mb genome sequence of Escherichia coli is also available. The life styles of the two bacteria are very different - H. influenzae is an obligate parasite that lives in human upper respiratory mucosa and can be cultivated only on rich media, whereas E. coli is a saprophyte that can grow on minimal media. A detailed comparison of the protein products encoded by these two genomes is expected to provide valuable insights into bacterial cell physiology and genome evolution. RESULTS: We describe the results of computer analysis of the amino-acid sequences of 1703 putative proteins encoded by the complete genome of H. influenzae. We detected sequence similarity to proteins in current databases for 92 % of the H. influenzae protein sequences, and at least a general functional prediction was possible for 83 %. A comparison of the H. influenzae protein sequences with those of 3010 proteins encoded by the sequenced 75 % of the E. coli genome revealed 1128 pairs of apparent orthologs, with an average of 59 % identity. In contrast to the high similarity between orthologs, the genome organization and the functional repertoire of genes in the two bacteria were remarkably different. The smaller genome size of H. influenzae is explained, to a large extent, by a reduction in the number of paralogous genes. There was no long range colinearity between the E. coli and H. influenzae gene orders, but over 70 % of the orthologous genes were found in short conserved strings, only about half of which were operons in E. coli. Superposition of the H. influenzae enzyme repertoire upon the known E. coli metabolic pathways allowed us to reconstruct similar and alternative pathways in H. influenzae and provides an explanation for the known nutritional requirements. CONCLUSIONS: By comparing proteins encoded by the two bacterial genomes, we have shown that extensive gene shuffling and variation in the extent of gene paralogy are major trends in bacterial evolution; this comparison has also allowed us to deduce crucial aspects of the largely uncharacterized metabolism of H. influenzae.

Bacterial Proteins↗
PubMed1996

Two domains of superfamily I helicases may exist as separate proteins.

DNA and RNA helicases of superfamily I are characterized by seven conserved motifs. The five N-terminal motifs are separated from the two C-terminal ones by a spacer that is highly variable in both sequence and length, suggesting the existence of two distinct domains. Using computer methods for protein sequence analysis, we show that PhoH, an ATP-binding protein that is conserved in Escherichia coli and Mycobacterium leprae, is homologous to the putative N-terminal domain of the helicases, whereas the putative E. coli protein YjhR is homologous to the C-terminal domain. These findings suggest that the N-and C-terminal domains of superfamily I helicases have distinct activities, with only the N-terminal domain having the ATPase activity. It is speculated that PhoH and YjhR have evolved from helicases through deletion of the portions of the helicase genes coding for the C- and N-terminal domain, respectively.

Amino Acid Sequence↗
PubMed1996

Protein sequence comparison at genome scale.

An adequate set of computer procedures tailored to address the task of genome-scale analysis of protein sequences will greatly increase the beneficial impact of the genome sequencing projects on the progress of biological research. This is especially pertinent given the fact that, for model organisms, one-half or more of the putative gene products have not been functionally characterized. Here we described several programs that may comprise the core of such a set and their application to the analysis of about 3000 proteins comprising 75% of the E. coli gene products. We find that the protein sequences encoded in this model genome are a rich source of information, with biologically relevant similarities detected for more than 80% of them. In the majority of cases, these similarities become evident directly from the results of BLAST searches. However, methods for motif analysis provide for a significant increase in search sensitivity and are particularly important for the detection of ancient conserved regions. As a result of sequence similarity analysis, generalized functional predictions can be made for the majority of uncharacterized ORF products, allowing efficient focusing of experimental effort. Clustering of the E. coli proteins on the basis of sequence similarity shows that almost one-half of the bacterial proteins have at least one paralog and that the likelihood that a protein belongs to a small or a large cluster depends on the function of this particular protein.

Algorithms↗
PubMed1996

Immunochemical structure of the OmpD porin from Salmonella typhimurium.

The OmpD porin was isolated and purified from Salmonella typhimurium strain SH 7454 (ompC::Tn10), digested with cyanogen bromide (CNBr) and the peptide fragments were separated by SDS-PAGE. N-terminal sequencing identified a total of 96 residues from four distinct peptides. The sequence showed that OmpD is homologous to NmpC (75% identity), Lc(75%) and OmpC (70%) from Escherichia coli, and OmpC (68%) from S. typhimurium. The sequence was essentially identical to the translated sequence of an nmpC-like gene of S. typhimurium, currently placed at 38.6 centisomes of the chromosome. Our results and other data suggest, however, that this gene is actually the ompD gene, which is more correctly placed in the 34 centisome region of the chromosome. The CNBr-generated peptides were also screened with 16 anti-S. typhimurium OmpD monoclonal antibodies by Western blotting. These results, in conjunction with the prediction of the OmpD folding pattern based on the known three-dimensional structure of E. coli OmpF, showed a close immunological relationship among S. typhimurium OmpD and E. coli NmpC and Lc, and a strong conservation of sequences within the transmembrane beta strands of these porins and E. coli OmpC, PhoE and OmpF, and Salmonella typhic OmpC.

Amino Acid Sequence↗
PubMed1995

Sequence similarity analysis of Escherichia coli proteins: functional and evolutionary implications.

A computer analysis of 2328 protein sequences comprising about 60% of the Escherichia coli gene products was performed using methods for database screening with individual sequences and alignment blocks. A high fraction of E. coli proteins--86%--shows significant sequence similarity to other proteins in current databases; about 70% show conservation at least at the level of distantly related bacteria, and about 40% contain ancient conserved regions (ACRs) shared with eukaryotic or Archaeal proteins. For > 90% of the E. coli proteins, either functional information or sequence similarity, or both, are available. Forty-six percent of the E. coli proteins belong to 299 clusters of paralogs (intraspecies homologs) defined on the basis of pairwise similarity. Another 10% could be included in 70 superclusters using motif detection methods. The majority of the clusters contain only two to four members. In contrast, nearly 25% of all E. coli proteins belong to the four largest superclusters--namely, permeases, ATPases and GTPases with the conserved "Walker-type" motif, helix-turn-helix regulatory proteins, and NAD(FAD)-binding proteins. We conclude that bacterial protein sequences generally are highly conserved in evolution, with about 50% of all ACR-containing protein families represented among the E. coli gene products. With the current sequence databases and methods of their screening, computer analysis yields useful information on the functions and evolutionary relationships of the vast majority of genes in a bacterial genome. Sequence similarity with E. coli proteins allows the prediction of functions for a number of important eukaryotic genes, including several whose products are implicated in human diseases.

Algorithms↗
PubMed1995

Detection of new genes in a bacterial genome using Markov models for three gene classes.

We further investigated the statistical features of the three classes of Escherichia coli genes that have been previously delineated by factorial correspondence analysis and dynamic clustering methods. A phased Markov model for a nucleotide sequence of each gene class was developed and employed for gene prediction using the GeneMark program. The protein-coding region prediction accuracy was determined for class-specific Markov models of different orders when the programs implementing these models were applied to gene sequences from the same or other classes. It is shown that at least two training sets and two program versions derived for different classes of E. coli genes are necessary in order to achieve a high accuracy of coding region prediction for uncharacterized sequences. Some annotated E. coli genes from Class I and Class III are shown to be spurious, whereas many open reading frames (ORFs) that have not been annotated in GenBank as genes are predicted to encode proteins. The amino acid sequences of the putative products of these ORFs initially did not show similarity to already known proteins. However, conserved regions have been identified in several of them by screening the latest entries in protein sequence databases and applying methods for motif search, while some other of these new genes have been identified in independent experiments.

Algorithms↗
PubMed1995

rpoE, the gene encoding the second heat-shock sigma factor, sigma E, in Escherichia coli.

In Escherichia coli, the heat shock response is under the control of two alternative sigma factors: sigma 32 and sigma E. The sigma 32-regulated response is well understood, whereas little is known about that of sigma E, except that it responds to extracytoplasmic immature outer membrane proteins. To further understand this response, we located the rpoE gene at 55.5' and analyzed the role of sigma E. sigma E is required at high temperature, and controls the transcription of at least 10 genes. Some of these might contribute to the integrity of the cell since delta rpoE cells are more sensitive to SDS plus EDTA and crystal violet. sigma E controls its own transcription from a sigma E-dependent promoter, indicating that rpoE transcription plays a role in the regulation of E sigma E activity. Indeed, under steady-state conditions, the transcription from this promoter mirrors the levels of E sigma E activity in the cell. However, it is unlikely that the rapid increase in E sigma E activity following induction can be accounted for solely by increased transcription of rpoE. Based upon homology arguments, we suggest that a gene encoding a negative regulator of sigma E activity is located immediately downstream of rpoE and may function as the target of the E sigma E inducing signal.

Amino Acid Sequence↗