PubMed Health⌕ Search

Biomedical subjects

F R Blattner

Publications and source records attributed to F R Blattner.

At least 55 records · Page 3Linked to original sources
PubMed1996

Improving the quality of automatic DNA sequence assembly using fluorescent trace-data classifications.

Virtually all large-scale sequencing projects use automatic sequence-assembly programs to aid in the determination of DNA sequences. The computer-generated assemblies required substantial hand-editing to transform them into submissions for GenBank. As the size of sequencing projects increases, it becomes essential to improve the quality of the automated assemblies so that this time consuming hand-editing may be reduced. Current ABI sequencing technology uses base calls made from fluorescently-labeled DNA fragments run on gels. We present a new representation for the fluorescent trace data associated with individual base calls. This representation can be used before, during, and after fragment assembly to improve the quality of assemblies. We demonstrate one such use-end-trimming of sub-optimal data-that results in a significant improvement in the quality of subsequent assemblies.

Algorithms↗
PubMed1995

Analysis of the Escherichia coli genome VI: DNA sequence of the region from 92.8 through 100 minutes.

The 338.5 kb of the Escherichia coli genome described here together with previously described segments bring the total of contiguous finished sequence of this genome to > 1 Mb. Of 319 open reading frames (ORFs) found in this 338.5 kb segment, 147 (46%) are potential new genes. The positions of several genes which had been previously located here by mapping or partial sequencing have been confirmed. Several ORFs have functions suggested by similarities to other characterised genes but cannot be assigned with certainty. Fifteen of the ORFs of unknown function had been previously sequenced. Eight transfer RNAs are encoded in the region and there are two grey holes in which no features were found. The attachment site for phage P4 and three insertion sequences were located. The region was also analysed for chi sites, bend sites, REP elements and other repeats. A computer search identified potential promoters and tentative transcription units were assigned. The occurrence of the rare tetramer CTAG was analysed in 1.6 Mb of contiguous E.coli sequence. Hypotheses addressing the rarity and distribution of CTAG are discussed.

Base Sequence↗
PubMed1995

Analogue of aspartate and glutamate active at synapses are attractants for Escherichia coli.

1. This research was carried out to compare Escherichia coli bacteria with animals in their response to L-aspartate and L-glutamate and their analogues. 2. Various analogues of aspartate and glutamate known to be neurotransmitters at synapses were shown to be attractants for E. coli. 3. The amino acid sequences of the animal receptors and the bacterial receptor, however, have no detectable relationship. Based on the amino acid sequence, evolutionarily the two systems appear not to be related.

Animals↗
PubMed1994

Analysis of the Escherichia coli genome. V. DNA sequence of the region from 76.0 to 81.5 minutes.

The DNA sequence of a 225.4 kilobase segment of the Escherichia coli K-12 genome is described here, from 76.0 to 81.5 minutes on the genetic map. This brings the total of contiguous sequence from the E.coli genome project to 725.1 kb (76.0 to 92.8 minutes). We found 191 putative coding genes (ORFs) of which 72 genes were previously known, and 110 of which remain unidentified despite literature and similarity searches. Seven new genes--arsE, arsF, arsG, treF, xylR, xylG, and xylH--were identified as well as the previously mapped pit and dctA genes. The arrangement of proposed genes relative to possible promoters and terminators suggests 90 potential transcription units. Other features include 19 REP elements, 95 computer-predicted bends, 50 Chi sites, and one grey hole. Thirty-one putative signal peptides were found, including those of thirteen known membrane or periplasmic proteins. One tRNA gene (proK) and two insertion sequences (IS5 and IS150) are located in this segment. The genes in this region are organized with equal numbers oriented with or against replication.

Alcohol Dehydrogenase↗
PubMed1994

A large-conductance mechanosensitive channel in E. coli encoded by mscL alone.

All cellular organisms respond to vibration, touch, gravity or changes in osmolarity, although the molecules on which such mechanosensations depend are unknown. Candidates include certain channels that gate in response to membrane stretch. Patch-clamp experiments with Escherichia coli envelope have revealed a mechanosensitive channel with very large conductance (MscL) and one with a smaller conductance (MscS) which may be important in osmoregulation. Here we have solubilized and fractionated the envelope, reconstituted the MscL activity in vitro, and traced it to a small protein, whose gene, mscL, we then cloned. Insertional disruption of mscL removes the channel activity, whereas re-expression of mscL borne on an expression plasmid restores it. MscL-channel activities were observed in material from a cell-free expression system with mscL as the only template. The mscL nucleotide sequence predicts a unique protein of only 136 amino acids, with a highly hydrophobic core and very different from porins or other known proteins.

Amino Acid Sequence↗
PubMed1993

Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli.

Sequences of four new heat-shock (HS) genes of Escherichia coli organized into two operons were determined. The operon at 83 min specifies two proteins of 15.8 kDa (HslT) and 16.1 kDa (HslS), which are identical to IbpA and IbpB, respectively. Expression of mRNA from a sigma 32-dependent promoter of the hslTS/ibpAB operon is stimulated 30-75-fold upon temperature upshift. The transcription start point (tsp) is located at a G, 96 bp upstream from the AUG start codon of hslT/ibpA. The deduced amino acid sequences of HslT/IbpA and HslS/IbpB are 48% identical to each other and were found to be remotely related to the chloroplast low-molecular-weight HS protein, which is highly conserved among plants. The second hs operon is much less actively stimulated by temperature upshift, although it has a hs promoter that perfectly matches the consensus of promoters recognized by sigma 32. Located at 88.9 min, the hslVU operon specifies proteins of 19.1 kDa (HslV) and 49.6 kDa (HslU). Multiple tsp were found in this operon. HslV is remotely related to the eukaryotic proteasome proteins, and HslU is very similar to a Pasteurella haemolytica protein of unknown function. Both HslU and the P. haemolytica protein share a ATP/GTP-binding motif near their N-termini. The two operons described here are transcribed counterclockwise on the standard genetic map.

ATP-Dependent Proteases↗
PubMed1993

Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes.

We present the sequence of 176 kilobases of the Escherichia coli K-12 genome, from katG at 89.2 to an open reading frame (ORF) of unknown function at 92.8 minutes on the genetic map. This brings the total of contiguous sequence from the E. coli genome project to 500 kb (81.5 to 92.8 minutes). This segment contains 134 putative coding genes (ORFs) of which 66 genes were previously identified. Eight new genes--acs, pepE, and nrfB-G--were identified as well as the previously mapped gldA and alr genes. Still, 58 ORFs remain unidentified despite literature and similarity searches. The arrangement of proposed genes relative to possible promoters and terminators suggests 55 potential transcription units. Other features include 13 REP elements, one IRU (ERIC) repeat, 59 computer-predicted bends, 42 Chi sites and one new grey hole. Sixteen signal peptides were found, including those of lamB, btuB, and malE. Two ribosomal RNA loci, rrnB and rrnE, are located in this segment, so we have now sequenced four of the seven E. coli rRNA loci. Comparison of the rRNA loci reveals some differences in the ribosomal structural RNAs which are generally compatible with the proposed secondary structures.

Base Sequence↗
PubMed1993

Genome sequencing on both strands: the Janus strategy.

The design of large scale DNA sequencing projects such as genome analysis demands a new approach to sequencing strategy, since neither a purely random nor a purely directed method is satisfactory. We have developed a strategy that combines these two methods in a way that preserves the advantages of both while avoiding their particular limitations. Computer simulations showed that a specific balance of random and directed sequencing was required for the most efficient strategy, termed the Janus strategy, which has been used in the Escherichia coli genome sequencing project. This approach depended on obtaining sequence easily from either strand of a cloned insert, and was facilitated by inversion of the insert in the engineered M13 vector Janus, by site-specific recombination. The inversion was accomplished simply by growth on the appropriate host strain, when the DNA strand incorporated into the new single stranded phage was complementary to that in the original phage, and was sequenced by the same simple protocol as the first strand.

Bacteriophage lambda↗
PubMed1993

Analysis of the Escherichia coli genome. III. DNA sequence of the region from 87.2 to 89.2 minutes.

The DNA sequence of 96.5 kilobases of the Escherichia coli K-12 genome has been determined, spanning the region between rrnA at 87.2 minutes and katG at 89.2 minutes on the genetic map. The sequence includes 84 open reading frames, of which 46 code for unidentified proteins. Six previously mapped but unsequenced genes have been identified in this span: mob, fdhD, rhaD, rhaA, rhaB, and kdgT. In addition, five new genes have been assigned: the heat shock genes hsIU and hsIV, and the genes fdoG, fdoH, and fdoI, which encode the three subunits of formate dehydrogenase-O. The arrangement of the genes relative to possible promoters and terminators suggests 57 potential transcription units. Other features include the precise location of the bacteriophage P2 attachment site attP2II, and eleven REP elements, including one containing 9 REP sequences--one of the largest such elements known. This segment brings the total length of contiguous finished sequence to 325 kilobases.

Base Sequence↗
PubMed1993

DNA sequence and analysis of 136 kilobases of the Escherichia coli genome: organizational symmetry around the origin of replication.

The DNA sequence of a 136-kb segment (81.5 to 84.5 min) from the Escherichia coli origin of replication region has been determined and analyzed. Of the 122 protein coding regions that were found, we could assign no gene name or function to half of them, even in this well-studied part of the genome. The newly sequenced region also includes five RNA genes. The arrangement of open reading frames and potential promoters suggests 63 transcription units. The sequence was also analyzed for bend sites and two types of repeated sequence elements. Together with our sequence of the 84.5 to 86.5 min region, this new determination forms a 227-kb contiguous region centered on oriC. A global analysis of this region reveals a remarkable symmetry: most genes are transcribed divergently from the replication origin, and Chi octanucleotide (5'GCTGGTGG3') recombinational hot spots are also strikingly oriented with respect to the directions of replication and translation.

Amino Acid Sequence↗
PubMed1993

Characterization of twenty-six new heat shock genes of Escherichia coli.

Most organisms respond to heat by substantial alteration of the pattern of gene expression. This has been particularly well studied with Escherichia coli although the response has by no means been completely characterized. Here we report the characterization of 26 new heat shock genes of E. coli, termed hsl, discovered by global transcription analysis with an overlapping lambda clone bank. We have measured the molecular weights of the corresponding heat shock proteins and mapped each of them to within a few kilobases on the E. coli genome. In vitro, 16 of them can be activated by the E sigma 32 RNA polymerase, which specifically transcribes heat shock genes. In vivo expression kinetics of seven of eight examined new proteins were found to be similar to those of the four most studied heat shock proteins, DnaK, DnaJ, GroEL (MopA), and GroES (MopB). In the course of this work, we confirmed that the catalytic subunit of the ATP-dependent Clp protease (also known as Ti protease), ClpP, is derived from a larger precursor protein. Possible assignments of some of the hsl genes to known proteins are discussed.

Bacterial Proteins↗
PubMed1993

Global regulation of gene expression in Escherichia coli.

Global transcription responses of Escherichia coli to various stimuli or genetic defects were studied by measuring mRNA levels in about 400 segments of the genome. Measuring mRNA levels was done by analyzing hybridization to DNA dot blots made with overlapping lambda clones spanning the genome of E. coli K-12. Conditions examined included isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, heat shock, osmotic shock, starvation for various nutrients, entrance of cells into the stationary phase of growth, anaerobic growth in a tube, growth in the gnotobiotic mouse gut, and effects of pleiotropic mutations rpoH, himA, topA, and crp. Most mapped genes known to be regulated by a particular situation were successfully detected. In addition, many chromosomal regions containing no previously known regulated genes were discovered that responded to various stimuli. This new method for studying globally regulated genetic systems in E. coli combines detection, cloning, and physical mapping of a battery of coregulated genes in one step.

Adaptation, Physiological↗
PubMed1992

Recombinant antibody-metallothionein: design and evaluation for radioimmunoimaging.

We have produced a chimeric antibody (Ab) in which metallothionein, a well-characterized biological chelator of metals, was genetically fused to the F(ab') domain of the S107 Ab heavy chain. Coexpression with the Ab light chain that conveys specificity for the synthetic antigen phosphocholine was achieved in plasmacytoma cells. Metal- and antigen-binding domains of the Ab-metallothionein hybrid function with normal avidity and specificity. Ab-metallothionein can be efficiently loaded with 99mTc and used to specifically bind phosphocholine-haptenated cells in vitro or to localize plasma-cell ascites tumors in mice. The approach offers potential advantages for producing radiolabeled Ab for targeted radiotherapy and diagnostic imaging.

Animals↗
PubMed1992

Metal-binding chimeric antibodies expressed in Escherichia coli.

Metallothionein, a well-characterized biological chelator of metals, has been genetically fused to the binding domain of an antibody and expressed in the periplasm of Escherichia coli. Specific delivery of 109Cd to immobilized hapten or to haptenated cells was demonstrated directly in periplasmic extracts. This approach is potentially useful for targeted radiotherapy and diagnostic imaging. We find six to seven atoms of metal per active antigen-combining site. Absence of the Fc portion of the immunoglobulin along with low immunogenicity of metallothionein-metal complexes should reduce immunologic reactions.

Amino Acid Sequence↗
PubMed1992

Analysis of the Escherichia coli genome: DNA sequence of the region from 84.5 to 86.5 minutes.

The DNA sequence of 91.4 kilobases of the Escherichia coli K-12 genome, spanning the region between rrnC at 84.5 minutes and rrnA at 86.5 minutes on the genetic map (85 to 87 percent on the physical map), is described. Analysis of this sequence identified 82 potential coding regions (open reading frames) covering 84 percent of the sequenced interval. The arrangement of these open reading frames, together with the consensus promoter sequences and terminator-like sequences found by computer searches, made it possible to assign them to proposed transcriptional units. More than half the open reading frames correlated with known genes or functions suggested by similarity to other sequences. Those remaining encode still unidentified proteins. The sequenced region also contains several RNA genes and two types of repeated sequence elements were found. Intergenic regions include three "gray holes," 0.6 to 0.8 kilobases, with no recognizable functions.

Bacterial Proteins↗
PubMed1991

Rapid detection of antigen binding by antibody fragments expressed in the periplasm of Escherichia coli.

Bacterial expression systems can greatly facilitate protein engineering of antibodies. We have developed a system for high-level expression of antibodies, antibody fragments, or hybrid antibodies with novel effector functions in the periplasm of Escherichia coli. From 5 ml of cells, a simple extraction yields sufficient material for SDS-gel electrophoresis, detection and characterization of hapten binding. To demonstrate our system, heavy-chain variable regions and lambda 1 light chains of a mouse anti-NP antibody were synthesized as hybrid proteins with a bacterial signal peptide (Omp F). Each chain is secreted into the periplasm where processing (cleavage of the signal peptide), folding and heterodimer association take place. Periplasmic proteins are released by cold osmotic shock, and hapten-binding activity is easily detected without further manipulation. The ease of genetic engineering in this system will facilitate the production of immunoglobulin derivatives designed for specific applications, and expression of these molecules in a native state will allow the rapid screening of combinatorial libraries and the results of mutagenesis.

Animals↗