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At least 613 records · Page 34Linked to original sources
PubMed1975

The predicted secondary structure of the N-terminal sequence of the lac repressor and proposed models for its complexation to the lac operator.

Rules for the prediction of protein conformation (Chou, P. Y., and Fasman, G. D. (1974), Biochemistry 13, 211, 222) have been applied to the N-terminal sequence 1-60 of the lac repressor. This analysis predicts beta structure at sequences 4-9 and 15-20, helices at 26-32, 38-45, and 52-57, and beta turns at 48-51 and 14-17. Repressor mutants lacking operator binding capacity in which Pro replaces Ser-16 and Ala replaces Thr-19 (Weber, K., Platt, T., Ganem, D., and Miller, J. H. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 3624) have no effect on the prediction of beta structure at residues 15 to 20, which suggests that the polar side chains of Ser-16, Tyr-17, Gln-18, and Thr-19 participate in intermolecular hydrogen bonding with conplementary polar groups on the lac operator. The loss of operator binding capacity on replacement of Ala by Val at position 53 in the repressor results from a predicted secondary structural change from helix to beta structure for residues 52-57 which can be transmitted to the N-terminal sequence via a beta turn at residues 48-51. The basic residues at positions 33, 35, and 37 between the helical regions 26-32 and 38-45 probably bind to the phosphate groups on the operator on complexation. It is proposed that complex formation involves the interaction of either a beta structure (residues 15-20) or a right-hand twisted antiparallel beta-pleated sheet (residues 4-9 and 12-20) with operator DNA.

Amino Acid Sequence↗
PubMed1991

PCR walking from microdissection clone M54 identifies three exons from the human gene for the neural cell adhesion molecule L1 (CAM-L1).

Microdissection has proved to be a powerful tool in the construction of libraries from specific chromosome segments (11) which are poorly covered by existing RFLP markers. Microclones also represent starting points for finding genes of interest. However, their length (100 to 200 bp) can make their use as probes problematic and identifying them as coding sequence is difficult. We report here that microclones can be extended in vitro by a modified version of our original PCR walking method (10) which utilises oligo-cassettes and the solid phase biotin/streptavidin separation system. We have extended the microclone M54, derived by dissection from Xq27.2 to proximal Xq28 (12), in both directions for approximately 700 bp. Direct sequencing of these products revealed that M54 was located within an intron of the human gene encoding the neural cell adhesion molecule L1 (CAM-L1) which has been recently mapped to Xq28 (13). The extension of M54 also identified three exons of this gene. This information allowed subsequent amplification of a 2.4 kb cDNA molecule from fetal human brain mRNA which encodes most of human CAM-L1. Sequencing of this cDNA revealed a high degree of sequence conservation with the mouse homologue (14). This is the first description of extension of a human derived microclone by PCR mediated walking within total human genomic DNA. These results show that anonymous DNA sequences may be extended into coding or any sequence.

Animals↗
PubMed1981

The DNA sequence of the promoter-attenuator of the ilvGEDA operon of Salmonella typhimurium.

The isolation of a lambda gt . ilvGEDA . S.t. hybrid transducing phage has permitted the characterization of the promoter-attenuator region of the ilvGEDA operon of Salmonella typhimurium. In vitro transcription and Southern hybridization indicate that the promoter-attenuator resides on a 400 nucleotide Rsa I restriction fragment. DNA sequence analysis shows only seven base pair differences exist between the DNA sequence of the ilvGEDA promoter-attenuator of S. typhimurium and that previously published for Escherichia coli K12.

Base Sequence↗
PubMed1989

Synthesis and characterization of a recombinant fragment of human alpha-fetoprotein with antigenic selectivity versus albumin.

A DNA sequence coding for human alpha-fetoprotein amino acid sequence 38-119 was synthesized and cloned in a bacterial expression vector. The alpha-fetoprotein sequence was selected as the least homologous to albumin, since the two proteins have an overall amino acid identity of approximately 38%. A chimeric protein was obtained which was purified by preparative electrophoresis and characterized in its primary structure by fast atom bombardment mass spectometry. About 70% of the alpha-fetoprotein sequence was physically mapped and found to correspond to the amino acids encoded in the synthetic gene. The use of this recombinant protein allowed the selection of monoclonal antibodies recognizing both the recombinant fragment and native alpha-fetoprotein. These antibodies should allow the development of an immunoassay for alpha-fetoprotein with absolute selectivity versus albumin. This might result in more sensitive clinical determinations, avoiding the possibility of cross-reactions.

Albumins↗
PubMed2001

Enlarging the amino acid set of Escherichia coli by infiltration of the valine coding pathway.

Aminoacyl transfer RNA (tRNA) synthetases establish the rules of the genetic code by catalyzing the aminoacylation of tRNAs. For some synthetases, accuracy depends critically on an editing function at a site distinct from the aminoacylation site. Mutants of Escherichia coli that incorrectly charge tRNA(Val) with cysteine were selected after random mutagenesis of the whole chromosome. All mutations obtained were located in the editing site of valyl-tRNA synthetase. More than 20% of the valine in cellular proteins from such an editing mutant organism could be replaced with the noncanonical aminobutyrate, sterically similar to cysteine. Thus, the editing function may have played a central role in restricting the genetic code to 20 amino acids. Disabling this editing function offers a powerful approach for diversifying the chemical composition of proteins and for emulating evolutionary stages of ambiguous translation.

Alleles↗
PubMed1996

Posttranscriptional osmotic regulation of the sigma(s) subunit of RNA polymerase in Escherichia coli.

The sigma(s) subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of sigma(s) during the exponential phase of growth. Osmotic induction of rpoS can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine. Stimulation of rpoS translation and a change in the half-life of sigma(s) from 3 to 50 min both contribute to osmotic induction. Experiments with lacZ fusions inserted at different positions within the rpoS gene indicate that an element required for sigma(s) degradation is encoded between nucleotides 379 and 742 of the rpoS coding sequence.

Bacterial Proteins↗
PubMed1968

Spontaneous and ICR191-A-induced frameshift mutations in the A gene of Escherichia coli tryptophan synthetase.

Frameshift mutant trpA21 was isolated after ultraviolet treatment and frameshift mutant trpA540 after ICR191-A (an acridine derivative) treatment of wild-type Escherichia coli K-12. The A proteins of spontaneous and ICR191-A-induced partial revertants of these mutants contained altered amino acid sequences one residue shorter than the comparable sequence in the A protein of wild-type bacteria. The data support the conclusion that ICR191-A causes frameshift mutations. The findings further indicate that both base additions and deletions are elicited by ICR191-A treatment and that mutagenesis by this compound sometimes affects more than one base pair. ICR191-A also weakly reverts some missense mutants. Analyses of the relevant peptides of the purified A protein show single amino acid replacements compatible with single base-pair changes. In addition, we found that some spontaneously revertible ICR191-A- and ultraviolet light-induced frameshift mutants are not further stimulated to revert by exposure to ultraviolet light.

Acridines↗
PubMed2001

DNA asymmetry and the replicational mutational pressure.

The mode of replication and organisation of bacterial genomes impose asymmetry on their nucleotide composition. The asymmetry is seen in coding and non-coding sequences and is reflected in the amino acid composition of proteins. The mechanisms generating asymmetry include: unequal mutation rates connected with replication and transcription, selection forces positioning genes and signal sequences nonrandomly in the genome, and protein coding constraints on coding sequences. There are different methods of visualising and measuring the asymmetry. Some of them can assess the contribution of individual mechanisms to the observed asymmetry and those have been described in greater detail. Asymmetric mutational and selection pressures differentiate the rates of evolution of genes on leading and lagging strands. The genes relocated to the opposite strand have to adapt to a different mutational pressure or are eliminated. Translocations from leading to lagging strands are more often selected against than from lagging to leading strands. Comparison of intergenic sequences that have lost the coding function to the original genes enables finding the frequencies of the twelve substitution rates in sequences free from selection. In the absence of selection, the half-time of substitution of a given type of nucleotide is linearly correlated with the fraction of that nucleotide in the sequence.

Journal Article↗
PubMed1987

Folding and activation of recombinant human prorenin.

In vitro folding of mature renin, prorenin, and fused prorenin, all produced in denatured form in inclusion bodies in recombinant Escherichia coli, has been studied in order to evaluate the importance of prosequence in the folding of human renin. These studies have been compared with the in vivo folding and subsequent in vitro activation of recombinant human prorenin secreted by a nonbacterial expression system, namely Chinese hamster ovary (CHO) cells grown in serum-free medium. It is concluded that prosequence is essential in the folding of human renin and, therefore, the DNA coding for this sequence cannot be removed without affecting the recovery of active human renin from recombinant bacterial and nonbacterial systems.

Cell Line↗
PubMed1987

Isolation and characterization of the yeast gene coding for the alpha subunit of mitochondrial phenylalanyl-tRNA synthetase.

The respiratory defect of pet mutants of Saccharomyces cerevisiae assigned to complementation group G120 has been ascribed to their inability to acylate the mitochondrial phenylalanyl tRNA. A fragment of wild type yeast genomic DNA capable of complementing the genetic lesion of G120 mutants has been cloned by transformation with a yeast genomic recombinant library of a representative mutant from this complementation group. The gene designated as MSF1 has been subcloned on a 2.2-kilobase pair fragment and its nucleotide sequence determined. The predicted protein product of MSF1 has a molecular weight of 55,314 and has several domains of high primary sequence homology to the alpha subunit of the Escherichia coli phenylalanyl-tRNA synthetase. Based on the phenotype of G120 mutants and the homology to the bacterial protein, MSF1 is proposed to code for the alpha subunit of yeast mitochondrial phenylalanyl-tRNA synthetase. Disruption of the chromosomal copy of MSF1 in the respiratory-competent haploid strain W303-1B induces a phenotype similar to G120 mutants but does not affect cell viability, indicating that the cytoplasmic phenylalanyl-tRNA synthetase of yeast is encoded by a separate gene. Although the E. coli and yeast mitochondrial aminoacyl-tRNA synthetases are sufficiently similar in their primary sequences to suggest a common evolutionary origin, they have undergone significant changes as evidenced by the low homology in some regions of the polypeptide chains and the presence in the mitochondrial enzyme of two domains that are lacking in the bacterial phenylalanyl-tRNA synthetase.

Amino Acid Sequence↗
PubMed1998

Genomic sequences of aldolase C (Zebrin II) direct lacZ expression exclusively in non-neuronal cells of transgenic mice.

Aldolase C is regarded as the brain-specific form of fructose-1, 6-bisphosphate aldolase whereas aldolase A is regarded as muscle-specific. In situ hybridization of mouse central nervous system using isozyme-specific probes revealed that aldolase A and C are expressed in complementary cell types. With the exception of cerebellar Purkinje cells, aldolase A mRNA is found in neurons; aldolase C message is detected in astrocytes, some cells of the pia mater, and Purkinje cells. We isolated aldolase C genomic clones that span the entire protein coding region from 1.5 kb 5' to the transcription start site to 0.5 kb 3' to the end of the last exon. The bacterial gene, lacZ, was inserted in two different locations and the constructs tested in transgenic mice. When the protein coding sequences were replaced with lacZ, three of five transgenic lines expressed beta-galactosidase only in cells of the pia mater; one line also expressed in astrocyte-like cells. When lacZ was inserted into the final exon (and all structural gene sequences were retained) transgene expression was observed in astrocytes in all regions of the central nervous system as well as in pial cells. Thus, with the exception of Purkinje cell expression, the behavior of the full-length transgene mimics the endogenous aldolase C gene. The results with the shorter transgene suggest that additional enhancer elements exist within the intragenic sequences. The absence of Purkinje cell staining suggests that the cis elements required for this expression must be located outside of the sequences used in this study.

Animals↗
PubMed2000

The ChiA (YheB) protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS.

The chromosome of Escherichia coli K-12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin. We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity. Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac- phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter. To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion-containing strain, selecting for a Lac+ phenotype. The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid-structuring protein H-NS. Transcription of chiA in vivo is driven by a single sigma70 promoter and is derepressed in an hns mutant. Using a competitive gel retardation assay, we demonstrated that H-NS binds directly and with high affinity to the chiA promoter region. In addition to hns, other E. coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37 degrees C to 30 degrees C. A possible physiological function of ChiA (YheB) endochitinase in E. coli K-12 is discussed.

Amino Acid Sequence↗
PubMed1991

The putative single-stranded DNA-binding protein of the filamentous bacteriophage, Ifl. Amino acid sequence of the protein and structure of the gene.

The protein product corresponding to the gene located in the region of the coliphage Ifl genome shown to contain the code for the single-stranded DNA (ssDNA)-binding proteins of all filamentous phages so far studied has been isolated from infected bacterial cells and its amino acid sequence determined. The mature protein contains 95 amino acids (calculated molecular mass 10553 Da). Its sequence corresponds to that predicted from the DNA sequence but lacks the initiating methionine residue. Although there is little direct sequence homology between the phage Ifl protein and the ssDNA-binding proteins of the other filamentous phages that have been studied, computer-based comparisons of various physical and structural parameters showed that the phage Ifl protein contains a domain that is closely related to domains in the coliphage T4 gene 32 protein and the Pseudomonas phage Pfl ssDNA-binding protein and suggest that the Ifl protein does have a ssDNA-binding function although we were unable to show this directly.

Amino Acid Sequence↗
PubMed2006

MBGD: a platform for microbial comparative genomics based on the automated construction of orthologous groups.

The microbial genome database for comparative analysis (MBGD) is a comprehensive platform for microbial comparative genomics. The central function of MBGD is to create orthologous groups among multiple genomes from precomputed all-against-all similarity relationships using the DomClust algorithm. The database now contains >300 published genomes and the number continues to grow. For researchers who are interested in ongoing genome projects, we have now started a new service called 'My MBGD,' which allows users to add their own genome sequences to MBGD for the purpose of identifying orthologs among both the new and the existing genomes. Furthermore, in order to make available the rapidly accumulating information on closely related genome sequences, we enhanced the interface for pairwise genome comparisons using the CGAT interface, which allows users to see nucleotide sequence alignments of non-coding as well as coding regions. MBGD is available at http://mbgd.genome.ad.jp/.

Algorithms↗
PubMed2006

A bacterial arginine-agmatine exchange transporter involved in extreme acid resistance.

The arginine-dependent extreme acid resistance response of Escherichia coli operates by decarboxylating arginine. AdiC, a membrane antiporter, catalyzes arginine influx coupled to efflux of the decarboxylation product agmatine, effectively exporting a proton in each turnover. Using the adiC coding sequence under control of a tetracycline promoter in an E. coli vector, we expressed and purified the transport-protein with a yield of approximately 10 mg/liter bacterial culture. Glutaraldehyde cross-linking experiments indicate that the protein is a homodimer in detergent micelles and lipid membranes. Purified AdiC reconstituted into liposomes exchanges arginine and agmatine in a strictly coupled, electrogenic fashion. Kinetic analysis yields K(m) approximately 80 microm for Arg, in the same range as its dissociation constant determined by isothermal titration calorimetry.

Agmatine↗