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PubMed1995

The diversity of alleles at the hsd locus in natural populations of Escherichia coli.

In enteric bacteria three discrete families of type I restriction and modification systems (IA, IB and ID) are encoded by alleles of the serB-linked hsd locus. Probes specific for each of the three families were used to monitor the distribution of related systems in 37 of the 72 wild-type Escherichia coli strains comprising the ECOR collection. All 25 members of group A in this collection were screened; 12 were probe-positive, nine have hsd genes in the IA family, two in the IB and one in the ID. Twelve strains, representing all groups other than A, were screened; five were probe-positive, one has hsd genes in the IA family, one in the IB and three in the ID. The type ID genes are the first representatives of this family in E. coli, the probe-negative strains could have alternative families of hsd genes. The type IA and IB systems added at least five new specificities to the five already identified in natural isolates of E. coli. The distribution of alleles is inconsistent with the dendrogram of the bacterial strains derived from other criteria. This discrepancy and the dissimilar coding sequences of allelic hsd genes both imply lateral transfer of hsd genes.

Alleles↗

PubMed1999

Compositional correlation studies among the three different codon positions in 12 bacterial genomes.

Compositional distributions in the three codon positions of the coding sequences of 12 fully sequenced prokaryotic genomes, which are publicly available, were investigated. A universal compositional correlation was observed in most of the genomes under investigation irrespective of their overall genomic GC contents. In all the genomes, the GC contents at the first codon positions are always greater than the overall GC contents of the genomes whereas the reverse is true in the case of second codon positions. GC contents at the third codon positions are higher than the overall genomic GC contents in high GC containing genomes, and the opposite situation was found in case of low GC genomes except for Helicobacter pylori. In high-GC rich genomes, the GC contents at the first + second codon positions are less than the GC contents at the third codon positions, and they are low in low-GC genomes except for Helicobacter pylori. The distributions of four bases at the three different positions were also investigated for all 12 organisms. It was observed that in high-GC genomes G is the most dominant base and in low-GC genomes A is the most dominant base in the first codon positions. But purine bases, i.e., (A + G), predominantly occur in the first codon position. In the second codon position, A is the most dominant base in most of the organisms and G is the least dominant base in all the organisms. There is no unique regular pattern of individual bases at the third codon positions; however, there are significant differences in the occurrences of (G + C) contents in the third codon positions among the different organisms. Calculations of dinucleotide frequencies in 12 different organisms indicate that in GC-rich genomes GG, GC, CC, and CG dinucleotides are the most dominant whereas the reverse is true in case of low-GC genomes. Biological implications of these results are discussed in this paper.

Bacteria↗

PubMed1992

Molecular and functional characterization of the murine glucocerebrosidase gene.

A genomic clone of glucocerebrosidase (D-glucosyl-N-acyl-sphingosine glucohydrolase; E.C. 3.2.1.45) purified from a genomic library derived from a Balb/c mouse was analyzed by restriction mapping and nucleotide sequencing of its promoter and protein coding regions. Promoter activity was functionally assessed by ligation of a 2 kb glucocerebrosidase fragment to the protein coding segment of a bacterial neomycin resistance gene. Smaller segments of the 5' flanking sequence were then analyzed for their ability to initiate transcription of the chloramphenicol acetyltransferase reporter gene. A 319 bp Eco RI-Bgl II fragment (containing 259 bp upstream of the cDNA 5' limit) ligated to the chloramphenicol acetyltransferase open reading frame produced considerable activity.

3T3 Cells↗

PubMed1981

The promoter-proximal region of the Bacillus licheniformis penicillinase gene: Nucleotide sequence and predicted leader peptide sequence.

Penicillinase (beta-lactamase) is a major species of secreted protein produced by Bacillus licheniformis 749. From the pTB2 recombinant plasmid containing the cloned entire penicillinase (penP) gene, we have isolated and sequenced a 446-bp HpaII fragment carrying the beginning of penP. The 3'-end coding region of 216-bp on this DNA fragment codes for the first 72 amino acids of the prepenicillinase protein. The deduced structure of the leader peptide consists of a 34 amino acid signal sequence with a hydrophilic N-terminal region and a central hydrophobic core.

Bacillus↗

PubMed2006

Vibrio cholerae CTXPhi phage repressor gene is borne on an insertion sequence-like element and encodes a putative transposase.

Bioinformatics analyses show the presence of a novel insertion sequence-like element in the CTXPhi phage of Vibrio cholerae. The solitary open reading frame encompassed by the element is known to encode a repressor (RstR) of phage DNA replication and is responsible for phage heteroimmunity. Analysis of the nucleotide and protein sequence of the repressor and its flanking non-coding regions indicates that it resembles distinctly simple bacterial insertion elements in numerous aspects. Based on the results of extensive sequence analysis, we propose that the rstR gene is borne on an insertion sequence-like element and that the RstR protein encodes a putative transposase. We put forward the possibility that a bi-directional promoter could regulate the divergent transcription of the neighbouring genes of rstR and rstA. The possibility of overlapping reading frames yielding distinct repressor and transposase proteins is also expanded based on the analyses.

Amino Acid Sequence↗

PubMed2005

An analysis of the use of genomic DNA as a universal reference in two channel DNA microarrays.

BACKGROUND: DNA microarray is an invaluable tool for gene expression explorations. In the two-dye microarray, fluorescence intensities of two samples, each labeled with a different dye, are compared after hybridization. To compare a large number of samples, the 'reference design' is widely used, in which all RNA samples are hybridized to a common reference. Genomic DNA is an attractive candidate for use as a universal reference, especially for bacterial systems with a low percentage of non-coding sequences. However, genomic DNA, comprising of both the sense and anti-sense strands, is unlike the single stranded cDNA usually used in microarray hybridizations. The presence of the antisense strand in the 'reference' leads to reactions between complementary labeled strands in solution and may cause the assay result to deviate from true values. RESULTS: We have developed a mathematical model to predict the validity of using genomic DNA as a reference in the microarray assay. The model predicts that the assay can accurately estimate relative concentrations for a wide range of initial cDNA concentrations. Experimental results of DNA microarray assay using genomic DNA as a reference correlated well to those obtained by a direct hybridization between two cDNA samples. The model predicts that the initial concentrations of labeled genomic DNA strands and immobilized strands, and the hybridization time do not significantly affect the assay performance. At low values of the rate constant for hybridization between immobilized and mobile strands, the assay performance varies with the hybridization time and initial cDNA concentrations. For the case where a microarray with immobilized single strands is used, results from hybridizations using genomic DNA as a reference will correspond to true ratios under all conditions. CONCLUSION: Simulation using the mathematical model, and the experimental study presented here show the potential utility of microarray assays using genomic DNA as a reference. We conclude that the use of genomic DNA as reference DNA should greatly facilitate comparative transcriptome analysis.

DNA↗

PubMed2005

Predicting candidate genomic sequences that correspond to synthetic functional RNA motifs.

Riboswitches and RNA interference are important emerging mechanisms found in many organisms to control gene expression. To enhance our understanding of such RNA roles, finding small regulatory motifs in genomes presents a challenge on a wide scale. Many simple functional RNA motifs have been found by in vitro selection experiments, which produce synthetic target-binding aptamers as well as catalytic RNAs, including the hammerhead ribozyme. Motivated by the prediction of Piganeau and Schroeder [(2003) Chem. Biol., 10, 103-104] that synthetic RNAs may have natural counterparts, we develop and apply an efficient computational protocol for identifying aptamer-like motifs in genomes. We define motifs from the sequence and structural information of synthetic aptamers, search for sequences in genomes that will produce motif matches, and then evaluate the structural stability and statistical significance of the potential hits. Our application to aptamers for streptomycin, chloramphenicol, neomycin B and ATP identifies 37 candidate sequences (in coding and non-coding regions) that fold to the target aptamer structures in bacterial and archaeal genomes. Further energetic screening reveals that several candidates exhibit energetic properties and sequence conservation patterns that are characteristic of functional motifs. Besides providing candidates for experimental testing, our computational protocol offers an avenue for expanding natural RNA's functional repertoire.

Algorithms↗

PubMed1992

Demonstration of relaxin precursors in pregnant rat ovaries with antisera against bacterially expressed rat prorelaxin.

The existence of rat 18-kilodalton (kDa) prorelaxin, which has been postulated from the coding sequence of cloned cDNA and the results of cell-free translation studies, has been directly demonstrated in rat ovaries with antibodies against bacterially expressed rat prorelaxin. The peptide expressed in E. coli from a rat prorelaxin cDNA construct was comprised of the B- and A-chains of relaxin and a 105-amino acid connecting region. Immunoreactive bands of 18 and 16.5 kDa were shown in ovaries from day 20 pregnant rats. Partial amino acid sequence analysis of both peptides revealed that they had identical N-terminal sequences, corresponding to rat prorelaxin. Both 18- and 16.5-kDa bands were present only from midpregnancy until near term, when they declined sharply. These changes in the concentration of 18-kDa prorelaxin match changes in preprorelaxin mRNA levels, suggesting that relaxin synthesis is regulated at the transcriptional level and not by protein processing. Prorelaxin was transiently secreted by COS-1 cells transfected with preprorelaxin cDNA. Treatment of culture medium with trypsin resulted in the appearance of material corresponding in size to mature relaxin. Thus, correctly folded prorelaxin appears to be a suitable precursor for relaxin. The combined concentrations of 18- and 16.5-kDa peptides in ovaries on day 20 of pregnancy were considerably more than 30 times greater than that of relaxin, however, suggesting that prorelaxin might also be more than a precursor per se.

Amino Acid Sequence↗

PubMed1997

Genome organization, natural genetic engineering and adaptive mutation.

Bacterial evolution is considered in the light of molecular discoveries about genome organization, biochemical mechanisms of genetic change, and cellular control networks. Prokaryotic genetic determinants are organized as modular composites of coding sequences and protein-factor binding sites joined together during evolution. Studies of genetic change have revealed the existence of biochemical functions capable of restructuring the bacterial genome at various levels and joining together different sequence elements. These natural genetic engineering systems can be subject to regulation by signal transduction networks conveying information about the extracellular and intracellular environments. Mu-mediated araB-lacZ coding sequence fusions provide one example of adaptive mutation (increased formation of useful mutations under selection) and illustrate how physiological regulation can modulate the activity of a natural genetic engineering system under specific conditions.

Bacteria↗

PubMed1995

Expression of active recombinant pallidipin, a novel platelet aggregation inhibitor, in the periplasm of Escherichia coli.

The platelet aggregation inhibitor pallidipin is a protein present in the saliva of the blood-sucking triatomine bug Triatoma pallidipennis. Expression of recombinant pallidipin in the periplasm of Escherichia coli was achieved by placing its coding sequence downstream of the alkaline phosphatase (APase) or trc promoter in frame with bacterial leader peptide DNA sequences derived from APase or from the periplasmic form of cyclophilin (Cph). In each case the DNA sequence of mature pallidipin was merged to the leader peptide coding part, either directly, or while introducing additional amino acids, in order to assess their influence on the activity of the leader peptidase and on the biological activity of the recombinant protein. All tested constructs gave rise to abundant periplasmic expression of pallidipin, which was then purified by a combination of cation- and anion-exchange chromatography followed by size-exclusion gel chromatography. Recombinant pallidipin had the expected molecular mass (approximately 19 kDa) and was correctly processed, as demonstrated by SDS/PAGE and N-terminal amino acid sequencing. The highest expression levels were obtained with the three APase-derived expression plasmids. Platelet aggregation tests revealed that E. coli-derived pallidipin was fully active, with an IC50 of 33-89 nM, comparable with that of the native protein, except when an additional N-terminal lysyl-isoleucyl dipeptide was present, which resulted in an IC50 more than ten times higher.

Amino Acid Sequence↗

PubMed2003

Characterization and expression of the Neurospora crassa nmt-1 gene.

The Neurospora crassa homologue of the yeast no message in thiamine ( nmt-1) gene was characterized. The deduced 342-amino-acid gene product has more than 60% identity with other fungal homologues and 42% similarity to a putative bacterial permease. In addition to three introns disrupting the coding sequence, a differentially spliced intron in the 5' untranslated region was also detected. Unlike other fungi, the N. crassa nmt-1 gene is repressed only 6- to 8-fold by exogenous thiamine concentrations above 0.5 microM; and a high basal level of nmt-1 mRNA persists even at 5 microM thiamine. Immuno-blotting with purified antibodies detected two variants of NMT-1 which differ in size and charge. The more abundant 39-kDa form is more strongly repressed by thiamine than the 37-kDa protein. NMT-1 abundance modulates slowly in response to changes in the concentration of exogenous thiamine, suggesting that N. crassa maintains thiamine reserves in excess of immediate needs. Disruption of the nmt-1 gene demonstrated that it is essential for growth in the absence of exogenous thiamine. NMT-1-deficient strains had a growth rate and colony density which was about 70% of the wild type, despite supplementation with a wide range of exogenous thiamine. These results suggest that the nmt-1 gene plays some other role in addition to thiamine biosynthesis.

Amino Acid Sequence↗

PubMed1989

Overexpression of native human beta 2-microglobulin in Escherichia coli and its purification.

beta 2-Microglobulin (beta 2M), the small subunit of human leukocyte antigen (HLA) class-I proteins, has been synthesized in Escherichia coli and purified in mg amounts. A beta 2m cDNA clone was fused in-frame behind DNA encoding the signal sequence for the outer membrane protein, OmpA. Three different constructions were made, whose products differed by the insertion of either an extra Ala residue, the hexapeptide AEFLEA [single-letter amino acid (aa) code], or no aa between the OmpA signal sequence and beta 2M-coding sequence. All three protein products were correctly processed by bacterial signal peptidase, as determined by N-terminal sequencing, and all three were secreted as soluble proteins into the periplasmic space. However, the signal sequence of the preprotein with the inserted hexapeptide, AEFLEA, was cleaved to a much greater degree than the other two preproteins. When there was no insertion, the mature protein was identical to human beta 2M, as analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, circular dichroism, and native isoelectric focusing. This 'bacterial beta 2M', radiolabeled with Bolton-Hunter reagent, was able to exchange into papain-solubilized HLA-B7, as determined by Sephadex G-75 chromatography and immune precipitation, indicating that bacterial beta 2M could complex with the heavy chain of HLA-B7.

Blotting, Western↗

PubMed1998

Susceptibility to recombination rearrangements of a chimeric plum pox potyvirus genome after insertion of a foreign gene.

Infectious RNA transcripts were generated from a chimeric cDNA clone of the plum pox potyvirus (PPV) genome containing the bacterial beta-glucuronidase (GUS) gene inserted between the sequences coding for the P1 and HC proteins. An artificial cleavage site specific for the NIa viral proteinase was engineered between the GUS and HC sequences to produce free GUS and HC proteins. The resulting virus PPVGus/ was stably maintained during the first round of infection, although plants remained symptomless and virus accumulation was delayed with respect to wild-type infection. PPVGus/ deleted variants, missing between 645 and 1779 nt, were detected in a subsequent plant passage. PPVGus/ deletions were confined inside the GUS gene, never affecting the P1 and HC coding regions, in contrast with previous reports of deletions in other potyvirus-based vector, in which deletions frequently reached the HC gene. These results suggest that the N-terminus of the PPV HC protein may be essential for virus viability. Analysis of the deletion endpoints showed short stretches of similarity in donor and acceptor RNAs, as well as oligo A tracts conserved in most junction sites, suggesting that deletions in PPVGus/ might take place by similarity-assisted recombination events.

Base Sequence↗

PubMed1997

Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants.

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1993

Molecular cloning and expression of an intracellular serpin: an elastase inhibitor from horse leucocytes.

Horse blood leucocytes contain an elastase inhibitor (HLEI) belonging to the serpin family. Poly(A)+RNA isolated from these cells was used to construct a cDNA library in lambda gt10, which was first screened with a synthetic degenerate oligonucleotide probe corresponding to the amino acid sequence of the reactive centre of the inhibitor. Three clones were obtained covering the entire coding region of the protein. Sequencing of these clones showed identity with the amino acid sequence obtained from Edman degradation of the elastase inhibitor. The coding sequence of the HLEI cDNA was cloned into the bacterial expression vector pKK233-2 and expressed in Escherichia coli cells. Transformed bacteria expressed significant amounts of the protein, which was immunoprecipitated with a specific anti-HLEI antiserum. Furthermore, HLEI expressed in bacteria inhibited the activity of elastase but not trypsin.

Amino Acid Sequence↗

PubMed1999

Cloning and characterization of human guanine deaminase. Purification and partial amino acid sequence of the mouse protein.

Mouse erythrocyte guanine deaminase has been purified to homogeneity. The native enzyme was dimeric, being comprised of two identical subunits of approximately 50,000 Da. The protein sequence was obtained from five cyanogen bromide cleavage products giving sequences ranging from 12 to 25 amino acids in length and corresponding to 99 residues. Basic Local Alignment Search Tool (BLAST) analysis of expressed sequence databases enabled the retrieval of a human expressed sequence tag cDNA clone highly homologous to one of the mouse peptide sequences. The presumed coding region of this clone was used to screen a human kidney cDNA library and secondarily to polymerase chain reaction-amplify the full-length coding sequence of the human brain cDNA corresponding to an open reading frame of 1365 nucleotides and encoding a protein of 51,040 Da. Comparison of the mouse peptide sequences with the inferred human protein sequence revealed 88 of 99 residues to be identical. The human coding sequence of the putative enzyme was subcloned into the bacterial expression vector pMAL-c2, expressed, purified, and characterized as having guanine deaminase activity with a Km for guanine of 9.5 +/- 1.7 microM. The protein shares a 9-residue motif with other aminohydrolases and amidohydrolases (PGX[VI]DXH[TVI]H) that has been shown to be ligated with heavy metal ions, commonly zinc. The purified recombinant guanine deaminase was found to contain approximately 1 atom of zinc per 51-kDa monomer.

Amino Acid Sequence↗

PubMed1998

Mutations in the liver glycogen phosphorylase gene (PYGL) underlying glycogenosis type VI.

Deficiency of glycogen phosphorylase in the liver gives rise to glycogen-storage disease type VI (Hers disease; MIM 232700). We report the identification of the first mutations in PYGL, the gene encoding the liver isoform of glycogen phosphorylase, in three patients with Hers disease. These are two splice-site mutations and two missense mutations. A mutation of the 5' splice-site consensus of intron 14 causes the retention of intron 14 and the utilization of two illegitimate 5' splice sites, whereas a mutation of the 3' splice-site consensus of intron 4 causes the skipping of exon 5. Two missense mutations, N338S and N376K, both cause nonconservative replacements of amino acids that are absolutely conserved even in yeast and bacterial phosphorylases. We also report corrections of the PYGL coding sequence, sequence polymorphisms, and a partial PYGL gene structure with introns in the same positions as in PYGM, the gene of the muscle isoform of phosphorylase. Our findings demonstrate that PYGL mutations cause Hers disease, and they may improve laboratory diagnosis of deficiencies of the liver phosphorylase system.

Amino Acid Sequence↗

PubMed1994

beta ig-h3: a transforming growth factor-beta-responsive gene encoding a secreted protein that inhibits cell attachment in vitro and suppresses the growth of CHO cells in nude mice.

beta ig-h3 is a novel gene first discovered by differential screening of a cDNA library made from A549 human lung adenocarcinoma cells treated with transforming growth factor-beta 1 (TGF-beta 1). It encodes a 683-amino-acid protein containing a secretory signal sequence and four homologous internal domains. Here we show that treatment of several types of cells, including human melanoma cells, human mammary epithelial cells, human keratinocytes, and human fibroblasts, with TGF-beta resulted in a significant increase in beta ig-h3 RNA. A portion of the beta ig-h3 coding sequence was expressed in bacteria, and antisera against the bacterially produced protein was raised in rabbits. This antisera was used to demonstrate that several cell lines secreted a 68-kD beta IG-H3 protein after treatment with TGF-beta. Transfection of beta IG-H3 expression plasmids into Chinese hamster ovary (CHO) cells led to a marked decrease in the ability of these cells to form tumors in nude mice. The beta IG-H3 protein was purified from media conditioned by recombinant CHO cells, characterized by immunoblotting and protein sequencing and shown to function in an anti-adhesion assay in that it inhibited the attachment of A549, HeLa, and WI-38 cells to plastic in serum-free media. Sequencing of cDNA clones encoding murine beta ig-H3 indicated 90.6% conservation at the amino acid level between the murine and human proteins. Finally, the beta ig-h3 gene was localized to human chromosome 5q31, a region frequently deleted in preleukemic myelodysplasia and leukemia. The corresponding mouse beta ig-h3 gene was mapped to mouse chromosome 13 region B to C1, which confirms a region of conservation on human chromosome 5 and mouse chromosome 13. We suggest that this protein be named p68 beta ig-h3.

Amino Acid Sequence↗