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PubMed2005

Sequence and comparative genomic analysis of actin-related proteins.

Actin-related proteins (ARPs) are key players in cytoskeleton activities and nuclear functions. Two complexes, ARP2/3 and ARP1/11, also known as dynactin, are implicated in actin dynamics and in microtubule-based trafficking, respectively. ARP4 to ARP9 are components of many chromatin-modulating complexes. Conventional actins and ARPs codefine a large family of homologous proteins, the actin superfamily, with a tertiary structure known as the actin fold. Because ARPs and actin share high sequence conservation, clear family definition requires distinct features to easily and systematically identify each subfamily. In this study we performed an in depth sequence and comparative genomic analysis of ARP subfamilies. A high-quality multiple alignment of approximately 700 complete protein sequences homologous to actin, including 148 ARP sequences, allowed us to extend the ARP classification to new organisms. Sequence alignments revealed conserved residues, motifs, and inserted sequence signatures to define each ARP subfamily. These discriminative characteristics allowed us to develop ARPAnno (http://bips.u-strasbg.fr/ARPAnno), a new web server dedicated to the annotation of ARP sequences. Analyses of sequence conservation among actins and ARPs highlight part of the actin fold and suggest interactions between ARPs and actin-binding proteins. Finally, analysis of ARP distribution across eukaryotic phyla emphasizes the central importance of nuclear ARPs, particularly the multifunctional ARP4.

Actins↗

PubMed1990

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.

Animals↗

PubMed1998

Changes in chromosomal localization of heterochromatin-binding proteins during the cell cycle in Drosophila.

We examined the heterochromatic binding of GAGA factor and proliferation disrupter (Prod) proteins during the cell cycle in Drosophila melanogaster and sibling species. GAGA factor binding to the brownDominant AG-rich satellite sequence insertion was seen at metaphase, however, no binding of GAGA factor to AG-rich sequences was observed at interphase in polytene or diploid nuclei. Comparable mitosis-specific binding was found for Prod protein to its target satellite in pericentric heterochromatin. At interphase, these proteins bind numerous dispersed sites in euchromatin, indicating that they move from euchromatin to heterochromatin and back every cell cycle. The presence of Prod in heterochromatin for a longer portion of the cell cycle than GAGA factor suggests that they cycle between euchromatin and heterochromatin independently. We propose that movement of GAGA factor and Prod from high affinity sites in euchromatin occurs upon condensation of metaphase chromosomes. Upon decondensation, GAGA factor and Prod shift from low affinity sites within satellite DNA back to euchromatic sites as a self-assembly process.

Adenine↗

PubMed1988

An RFLP adjacent to the maize waxy gene has the structure of a transposable element.

Two maize inbred lines harbor non-mutant waxy (Wx) genes that display restriction fragment length polymorphism (RFLP) upstream from the start of Wx transcription. Sequencing of this region in the two strains revealed a DNA insertion with the structural features of a transposable element. The insertion is 316 bp in length, has 15 bp imperfect inverted repeats and is flanked by a 5 bp direct repeat generated upon insertion. Sequences homologous to this insertion are present in multiple copies in maize and its relatives teosinte and Tripsacum but not in the more distantly related dicot tobacco. Finally, this element is not homologous with any previously described maize DNA insertion.

Base Sequence↗

PubMed1979

Mu insertion duplicates a 5 base pair sequence at the host inserted site.

Nucleotide sequences were analyzed across the two ends of lysogenic Mu DNA. These ends were cloned separately in lambdapMu hybrid particles that derived from a single Mu lysogen in the lac Z part of lambdaplac5. The obtained data imply that Mu lysogenization was associated with the duplication of 5 base pairs present in lac DNA at the Mu insertion site. As a result of this duplication, Mu DNA is flanked by two copies of five identical base pairs oriented as direct repeats. A similar conclusion has been obtained independently by other investigators with the use of a different Mu lysogen (D. Kamp and R. Kahmann, personal communication). Thus Mu insertion seems to have a striking similarity to typical IS-mediated insertions that were found to be associated with a short DNA duplication at the target site.

Base Sequence↗

PubMed2002

Regulation and adaptive evolution of lactose operon expression in Lactobacillus delbrueckii.

Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts, L. delbrueckii subsp. bulgaricus loses its ability to regulate lac operon expression. A series of mutations led to the constitutive expression of the lac genes. A complex of insertion sequence (IS) elements (ISL4 inside ISL5), inserted at the border of the lac promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A lac repressor gene was discovered downstream of the beta-galactosidase gene of L. delbrueckii subsp. lactis and was shown to be inactivated by several mutations in L. delbrueckii subsp. bulgaricus. Regulatory mechanisms of the lac gene expression of L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis were compared by heterologous expression in Lactococcus lactis of the two lac promoters in front of a reporter gene (beta-glucuronidase) in the presence or absence of the lac repressor gene. Insertion of the complex of IS elements in the lac promoter of L. delbrueckii subsp. bulgaricus increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the lac repressor by mutations was then necessary to induce the constitutive expression of the lac genes in L. delbrueckii subsp. bulgaricus.

Adaptation, Physiological↗

PubMed1998

The expression of the protective antigen of Bacillus anthracis in Bacillus subtilis.

The expression of Bacillus anthracis protective antigen (PA) in B. subtilis from the pag gene in pPA101-1 was explored in different genetic backgrounds in an attempt to identify opportunities to maximize expression. Introduction of AtxA, which positively regulates PA expression in B. anthracis did not improve expression levels in the protease-deficient strain WB600. Plasmid pPA101-1 was found to carry a deletion which created a new fusion point between vector and insert sequence, and which removed part of the AtxA binding site. The deletion may have occurred as a consequence of recombination between TCTAT sequences within both the vector and insert. Host mutations could influence expression; PA levels from pPA101-1 are threefold higher in a ccpA mutant than in an otherwise isogenic parent, and eightfold higher in an abrB mutant. These data demonstrate that the introduction of mutations affecting catabolite repression and growth phase regulation results in an increase in the yield of PA in this host-vector system. Combining these mutations with a multiply protease-negative background could potentially allow further improvements in PA yield.

Antigens, Bacterial↗

PubMed1999

Targeting of tail-anchored proteins to yeast mitochondria in vivo.

Tail-anchored proteins are inserted into intracellular membranes via a C-terminal transmembrane domain. The topology of the protein is such that insertion must occur post-translationally, since the insertion sequence is not available for membrane insertion until after translation of the tail-anchored polypeptide is completed. Here, we show that the targeting information in one such tail-anchored protein, translocase in the outer mitochondrial membrane 22, is contained in a short region flanking the transmembrane domain. An equivalent region is sufficient to specify the localisation of Bcl2 and SNARE proteins to the secretory membranes. We discuss the targeting process for directing members of this protein family to the secretory and mitochondrial membranes in vivo.

Amino Acid Sequence↗

PubMed2000

From a short amino acidic sequence to the complete gene.

A useful strategy directed to the isolation of a required gene with a high GC content is reported. Using a degenerate oligonucleotide probe, deduced from the amino terminus of a protein, it is possible to obtain a fragment of DNA containing its encoding gene by PCR amplification. Furthermore, the cloning of a desired gene can be accomplished in two steps by using an oligonucleotide deduced (i) from an internal sequence, (ii) from a consensus sequence, or (iii) from a DNA sequence adjacent to a disrupting element (transposon, insertion sequence, cassette). This method, which could be applied to a bacteriophage, plasmid, or cosmid genomic library, has been successfully used for cloning several genes from different biological systems.

Amino Acid Sequence↗

PubMed1999

Up-regulation of Eph tyrosine kinase receptors after excitotoxic injury in adult hippocampus.

The molecular mechanisms underlying the response to injury in the central nervous system are incompletely understood. Many cell activation systems may be involved. Tyrosine kinase receptors and their ligands play key roles in cell activation throughout life. The Eph family of tyrosine kinase receptors/ ligands are developmentally regulated and have been implicated in neural pathfinding. However, nothing is known about their role in the adult brain. We have used a model of central nervous system lesion in the rat, in which intraventricular injection of kainate was performed. This produced neuronal death in the CA3-CA4 fields and glial activation in the hippocampus. Highly degenerate primers, corresponding to the catalytic domain of the tyrosine kinase family, were used for reverse transcription-polymerase chain reaction of pooled RNA extracted from injured hippocampi. The amplified products were cloned and 100 clones (arbitrarily named TK1-TK100) were examined and inserts sequenced. We obtained four clones containing inserts which belong to the Eph receptor family. Two of these inserts (TK17 and TK63) were EphA4 and the other were EphB2 (TK25) and EphA5 (TK23). We performed in situ hybridization, and we found our clones to be present in all fields of the hippocampus, their expression being mainly neuronal. Three days after lesion, prominent expression appeared in CA1 as compared to the same field in the non-treated contralateral hippocampus. We performed northern blot analysis for quantification, and found that, three days after injury, the values decreased to 33 +/- 4%, 33 +/- 1% and 46 +/- 1% of control values for TK63 (EphA4), TK25 (EphB2) and TK23 (EphA5), respectively. Neuronal death in CA3-CA4 might account for this fact. Later, five days post-injury, the expression increased to 63 +/- 3%, 71 +/- 1% and 111 +/- 5% of control values, respectively. This increase was due to an up-regulation of these genes in the hippocampal neurons that survive after the injury, as indicated by in situ hybridization.

Amino Acid Sequence↗

PubMed2004

Selection and characterization of conditionally active promoters in Lactobacillus plantarum, using alanine racemase as a promoter probe.

This paper describes the use of the alr gene, encoding alanine racemase, as a promoter-screening tool for the identification of conditional promoters in Lactobacillus plantarum. Random fragments of the L. plantarum WCFS1 genome were cloned upstream of the promoterless alr gene of Lactococcus lactis in a low-copy-number plasmid vector. The resulting plasmid library was introduced into an L. plantarum Deltaalr strain (MD007), and 40,000 clones were selected. The genome coverage of the library was estimated to be 98%, based on nucleotide insert sequence and restriction analyses of the inserts of randomly selected clones. The library was screened for clones that were capable of complementing the D-alanine auxotroph phenotype of MD007 in media containing up to 10, 100, or 300 micro g of the competitive Alr inhibitor D-cycloserine per ml. Western blot analysis with polyclonal antibodies raised against lactococcal Alr revealed that the Alr production level required for growth increased in the presence of increasing concentrations of D-cycloserine, adding a quantitative factor to the primarily qualitative nature of the alr complementation screen. Screening of the alr complementation library for clones that could grow only in the presence of 0.8 M NaCl resulted in the identification of eight clones that upon Western blot analysis showed significantly higher Alr production under high-salt conditions than under low-salt conditions. These results established the effectiveness of the alanine racemase complementation screening method for the identification of promoters on their conditional or constitutive activity.

Alanine Racemase↗

PubMed1996

Chimeric probe-mediated ribonuclease protection assay for molecular diagnosis of mRNA deficiencies.

Investigations throughout the last decade have established that cytoskeleton integrity ensures red cell deformability and mechanical stability, and that defects in one of the skeletal components usually result in more or less severe hemolytic anemias. Although a large number of molecular defects have been identified to date, many others still bypass fast and commonly used methods, such as SSCP and DGGE, mostly because of a subtle change in mRNA transcription level or a complex interaction leading to the loss of other components. We describe a ribonuclease protection assay based on a simultaneous quantification of two cytoskeletal transcripts, using a chimeric probe, emphasizing the value of a nonspecific bridging sequence, inserted between the two specific probe sequences. It is anticipated that this powerful and reliable procedure would be an additional tool in the methodology array used for screening cytoskeletal inherited abnormalities.

Anion Exchange Protein 1, Erythrocyte↗

PubMed2000

Human membrane type-2 matrix metalloproteinase is defective in cell-associated activation of progelatinase A.

Transfection of the mouse membrane type-2 matrix metalloproteinase (MT2-MMP) gene into COS-1 cells resulted in activation of progelatinase A; however, that of the human gene had no effect. Expression of human and mouse MT2-MMP chimeric proteins revealed the defect of human MT2-MMP which resides in the region between amino acid (aa) residues 155 and 271. Seven aa residues in this region were not conserved between human and mouse MT2-MMP. Substitution with the corresponding mouse residue, proline-183 to serine and glutamine-185 to aspartic acid, recovered cell-associated progelatinase A activation function. These residues are located in the insertion sequence-2 (IS-2), which was conserved in six clones of the human MT2-MMP gene from different sources, except that of proline-183 which was substituted with serine from HT1080 cells. These results indicate that human MT2-MMP is defective in cell-associated activation of progelatinase A, and this is attributed to IS-2. These findings emphasize the importance of IS-2 in MT2-MMP functionality.

Amino Acid Sequence↗

PubMed1980

Transposition of IS1-lambdaBIO-IS1 from a bacteriophage lambda derivative carrying the IS1-cat-IS1 transposon (Tn9).

Tn9 is a transposable element in which a gene (cat) determining chloramphenicol resistance is flanked by directly repeated sequences that are homologous to the insertion sequence IS1. We show here that infection of Escherichia coli K12 (under Rec-Red-Int- conditions) with a lambda bio transducing phage carrying Tn9 results in the formation of lambda bio transductants as frequently as cat transductants as frequently as cat transductants (about 1 per 10(6) to 10(7) infected cells). Most of the lambda bio transductants do not carry cat, just as most of the cat transductants do not carry lambda bio. In spite of the absence of cat, the lambda bio prophage can transpose a second time, from the E. coli chromosome to different sites on an F'gal plasmid. Analysis of the structure of the transposed lambda bio element, by restriction nuclease digestion and by electron microscopy, demonstrates that the integrated lambda bio prophage is flanked by directly repeated IS1 elements. We conclude that there is no genetic information for the ability to transpose encoded in the non-repeated portion of Tn9, i.e. that the directly repeated IS1 elements alone are responsible for Tn9 transposition.

Bacteriophage lambda↗

PubMed1994

The chloroplast gene cluster containing psbF, psbL, petG and rps3 is conserved in Chlamydomonas.

We have sequenced a 6.8-kb segment of the Chlamydomonas eugametos chloroplast DNA which contains the psbF, psbL, petG and rps3 genes. As in the distantly related green alga Chlamydomonas reinhardtii, these genes reside in this order (5'-->3') on the same DNA strand, suggesting that such a chloroplast gene cluster was present in the most recent common ancestor of all Chlamydomonas species. For each of the four genes, with the exception of rps3, the C. eugametos and C. reinhardtii coding regions were found to be identical, or very similar, in length, whereas each of the intergenic spacers is substantially longer in C. eugametos than in C. reinhardtii. The central portion of both Chlamydomonas rps3 genes features a long extra coding region relative to other rps3 sequences. We have shown that the insertion sequence in the C. eugametos rps3 is not excised at the RNA level.

Amino Acid Sequence↗

PubMed1986

Identification and sequence analysis of a silent gene (ushA0) in Salmonella typhimurium.

The evolution of new or improved enzyme specificities in prokaryotes has been proposed to involve gene duplication, followed by silencing of one of the duplicates at the transcriptional or translational level. Such "silent gene intermediates" are distinct from "cryptic" genes, which are proposed to have a different role in evolution. We describe the identification in Salmonella typhimurium of a silent gene (ushA0) using the active (homologous) ushA gene (encoding UDP-sugar hydrolase) from Escherichia coli as a probe. The ushA0 gene has been cloned and, in the multicopy state, very weak expression can be detected; the gene product was shown to be immunologically and functionally related to the enzyme from E. coli. The sequence of the ushA0 gene was found to be highly homologous to the previously determined sequence of the ushA gene, and the respective promoter and ribosomal-binding sites are also very similar. However, a presumed strong rho-independent terminator in the ushA gene is absent from ushA0; although a weak stem-and-loop structure is present in the 3' region of ushA0, its structure is atypical of rho-independent terminators. The sequence analysis also revealed an insertion-sequence like sequence at the 3' end of ushA0 with a convergent open reading frame terminating 116 base-pairs from the ushA0 stop codon. A deletion of the 5' region of the open reading frame results in increased expression of ushA0, indicating that convergent transcription plays some role in the silencing of ushA0. S. typhimurium contains a UDP-sugar hydrolase, biochemically and genetically distinct from that in E. coli, encoded by the ushB gene. Our results indicate that ushB is not strongly sequence-related to ushA0, and its gene product is not immunologically related to the ushA gene product. ushB is hence a functional duplicate of ushA and provides a rationale for the silencing of ushA0. This situation, and the DNA sequence comparison of ushA and ushA0, strongly suggests that rather than being a cryptic gene, ushA0 has been silenced recently during the evolution of S. typhimurium.

Base Sequence↗

PubMed2003

IS900/ERIC-PCR as a tool to distinguish Mycobacterium avium subsp. paratuberculosis from closely related mycobacteria.

There is an increasing demand for fast and reliable methods to distinguish Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) from closely related mycobacteria and also a need for rapid strain specific typing of clinical isolates for epidemiological reasons. In the present study, the potential of rep-PCR as a fingerprinting method for M. paratuberculosis was assessed and compared to conventional RFLP. A PCR assay was designed and optimised to obtain reproducible fingerprints of mycobacterial DNA with primers targeting the enterobacterial intergenic consensus (ERIC) sequence and the M. paratuberculosis specific insertion sequence IS900. Reproducible fingerprints were obtained with 60 strains of M. paratuberculosis, 16 strains of M. avium subsp. avium, 3 strains of M. intracellulare, and 11 other mycobacterial strains. A species-specific band pattern that was clearly distinguishable from that of other mycobacteria was obtained with M. paratuberculosis. The rep-PCR did not detect any differences among M. paratuberculosis strains of different RFLP types, and was therefore not considered as an alternative fingerprinting method. However, the species-specific band pattern make IS900/ERIC-PCR a suitable alternative for distinguishing M. paratuberculosis from other mycobacteria, especially in cases of IS900 PCR positive mycobacteria. The fingerprinting method reported was fast and easy to perform, and produced highly reproducible results.

Animals↗

PubMed1997

A 'core ATPase', Hsp70-like structure is conserved in human, rat, and C. elegans STCH proteins.

We have identified the rat and Caenorhabditis elegans homologues of a 'core ATPase'-encoding Hsp70-like gene, designated Stch. We observed that the human, rat, and C. elegans Stch genes have conserved a stop codon immediately distal to the sequence encoding the Hsp70 ATPase domain. This results in the functional equivalent of an N-terminal, proteolytically cleaved fragment of Hsc70/BiP. Each homologue contains a hydrophobic signal sequence, demonstrates striking identity within the Hsp70 ATPase domain, and retains a similar C-terminal sequence (STCH specific cluster III) that is unique among Hsp70 proteins and which truncates the peptide binding domain. In addition, we have identified an internal 35-aa region that is homologous to the minimal sequence of the Hip chaperone co-factor that is required for direct binding to the ATPase domain of Hsp70. Adjacent to this region, the rat and human STCH protein sequences diverge within a short internal 'insertion' sequence that interrupts the ATPase subdomain between the phosphate-2 and adenosine ATP-binding sites. We have also demonstrated that both human and rat Stch are constitutively produced and are induced by the calcium ionophore A23187, but not by heat shock. The recognition that the truncated 'core ATPase' structure of the STCH molecule is conserved in human, rat, and C. elegans tissues suggests an important role for this unique member of the membrane-bound Hsp70 family.

Adenosine Triphosphatases↗