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PubMed1989

A synthetic strategy for simultaneous purification-conjugation of antigenic peptides.

A novel approach to the preparation of immunopeptide-carrier protein conjugates of improved chemical definition based on a solid-phase synthetic protocol combining incorporation of a Cys(Npys) N-terminal residue with systematic acetylation after every coupling, is described. The potential of the method is demonstrated in a synthesis of the tridecapeptide (Npys)-Cys-Val-Asn-Tyr-Ile-Arg-Lys-Arg-Ser-Leu-Gln-Thr-Val-OH in which the main product is purposely contaminated by a number of shorter truncated sequences resulting from intentionally defective couplings. From this peptide crude, and rather independently of its complexity, the target sequence can be selectively recovered and attached to a carrier molecule through a disulfide bond formed by reaction of the Npys-protected cysteine residue and a thiol function in the carrier. The process can be properly named purification-by-conjugation.

Amino Acid Sequence↗
PubMed1991

A common precursor for a putative hemorrhagic protein and rhodostomin, a platelet aggregation inhibitor of the venom of Calloselasma rhodostoma: molecular cloning and sequence analysis.

Rhodostomin is a platelet aggregation inhibitor secreted by the venom gland of Calloselasma rhodostoma. We report here the isolation of a 1.67-kilobase (kb) lambda gt11 cDNA clone using degenerate oligonucleotide probe based on a partial amino acid sequence of rhodostomin. The amino acid sequence deduced from an open reading frame of the cDNA indicates that (i) the 68-amino acid sequence of rhodostomin is located at the carboxyl terminus of the precursor polypeptide and (ii) a peroxisomal targeting sequence (ser.his.ala.) exists between the stop codon and the rhodostomin sequence of the precursor. Since the amino-terminal segment of the deduced sequence shows a high degree of identity with hemorrhagic proteins, which are zinc-metalloproteinases, found in the venom of some crotalid and viperid snakes, our results also predict the existence of at least one such hemorrhagic protein in the venom of Calloselasma rhodostoma. The derivation of a platelet aggregation inhibitor and a hemorrhagic protein from the same precursor protein is consistent with the fact that these proteins may be synergistic in function.

Amino Acid Sequence↗
PubMed1989

An assessment of optimal conditions for amplification of HIV cDNA using Thermus aquaticus polymerase.

Optimal conditions for in-vitro enzymatic DNA amplification using Thermus aquaticus polymerase were defined using env and gag sequences of human immunodeficiency virus type I as templates. It was found that specific amplification of the target sequence occurred when the primer annealing temperature was above 55 degrees C. Polymerase added once at the beginning of the procedure was sufficient for good results. Deoxynucleotide and primer concentrations and chain elongation time were not found to be important as long as they were kept above a critical level. Differences were noted between the efficiency of specific amplification from purified plasmid and genomic DNA. A rapid and simple method without the use of radioactive reagents for confirmation of a suggestive positive result on gel electrophoresis using restriction enzyme digestion of the amplified products is described. Some possible drawbacks of the technique particularly if used as a diagnostic tool are discussed.

Base Sequence↗
PubMed1996

Genomic organization of the fungus Phycomyces.

The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G + C) content, 35%; the coding sequences cloned to date all have a G + C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5'-CCGG-3') and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G + C-rich genes surrounded by G + C-poor areas. Two clones have similarities to the transposase of the transposon Tc1 from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tc1-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Tx1 from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 bp named PrA1 and PrA2.

Amino Acid Sequence↗
PubMed1990

Genus- and species-specific DNA probes to identify mycobacteria using the polymerase chain reaction.

Differential diagnosis of Mycobacterium tuberculosis, M. avium, and other mycobacteria remains a lengthy process. Recently, the use of DNA probes has been proposed as a new approach for a more specific and rapid diagnosis. Here, we report the cloning and sequencing of a genus-specific probe for Mycobacterium and a species-specific M. avium probe. The genus-specific probe hybridizes with DNA from nine ATCC type strains and 13 isolates of mycobacteria but not to non-mycobacterial DNA. In addition, the cloned fragment could also be amplified by polymerase chain reaction (PCR) in DNa of ten different mycobacterial type strains. The M. avium specific probe hybridizes strongly to sequences amplified in M. avium but not other mycobacterial or non-mycobacterial DNA. Amplification of the target sequence by PCR allowed the detection of 1 fg of all mycobacterial DNA tested for the genus-specific probe and 1 fg of M. avium DNA for the species-specific probe.

Animals↗
PubMed2005

A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor poils-au-dos, a repressor of achaete-scute.

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. Here, we describe a new gene involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos, that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null, alleles of poils au dos, is associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete-scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, we show that pad interacts synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively.

Alleles↗
PubMed2000

The piggyBac element is capable of precise excision and transposition in cells and embryos of the mosquito, Anopheles gambiae.

The piggyBac transposable element was tested for transposition activity in plasmid-based excision and inter-plasmid transposition assays to determine if this element would function in Anopheles gambiae cells and embryos. In the Mos55 cell line, precise excision of the piggyBac element was observed only in the presence of a helper plasmid. Excision occurred at a rate of 1 event per 1000 donor plasmids screened. Precise excision of the piggyBac element was also observed in injected An. gambiae embryos, but at a lower rate of 1 excision per 5000 donor plasmids. Transposition of the marked piggyBac element into a target plasmid occurred in An. gambiae cells at a rate of 1 transposition event per 24,000 donor plasmids. The piggyBac element transposed in a precise manner, with the TTAA target site being duplicated upon insertion, in 56% of transpositions observed, and only in the presence of the piggyBac helper. The remaining transpositions resulted in a deletion of target sequence, a novel observation for the phenomenon of piggyBac element insertion. 'Hot spots' for insertion into the target plasmid were observed, with 25 of 34 events involving one particular site. These results are the first demonstration of the precise mobility of piggyBac in this malaria vector and suggest that the lepidopteran piggyBac transposon is a candidate element for germline transformation of anopheline mosquitoes.

Animals↗
PubMed1997

Applications of transposition mutagenesis in antibiotic producing streptomycetes.

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the normally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.

Anti-Bacterial Agents↗
PubMed1998

cDNA cloning and expression analysis of genes encoding GSH synthesis in roots of the heavy-metal accumulator Brassica juncea L.: evidence for Cd-induction of a putative mitochondrial gamma-glutamylcysteine synthetase isoform.

In roots of Brassica juncea L. cadmium (Cd) exposure (25 microM) induces a massive formation of phytochelatins (PCs), which is accompanied by an only moderate decrease (-20%) of the putative PC precursor glutathione (GSH). As PC formation in roots could be the result of local GSH de novo synthesis and/or depend on GSH import from the shoot, we have analyzed the expression of the enzymes involved in GSH synthesis in the root, namely OAS(thiol)lyase (OAS-TL; catalysing the last step in Cys biosynthesis), gamma-glutamylcysteine synthetase (gamma-ECS), and glutathione synthetase (GSHS). cDNA clones were isolated from a cDNA library prepared from heavy metal exposed roots. Protein sequences from cDNA clones encoding OAS-TL, gamma-ECS, and GSHS, all exhibited putative mitochondrial targeting sequences, however, for OAS-TL also two putative cytosolic isoforms were isolated. Furthermore, we have cloned several metallothionein cDNAs of the MT2 group. Northern blot analysis with coding region probes revealed that in roots of Cd-exposed plants transcript amounts for OAS-TL and GSHS were only moderately increased, whereas gamma-ECS mRNA showed a stronger increase. Expression analysis with 3'-UTR probes indicated that among the putative mitochondrial OAS-TL, gamma-ECS and GSHS isoforms only gamma-ECS was up-regulated in response to Cd exposure. Conversely, transcripts for MT2 appeared to be slightly reduced. The results indicate that in roots Cd-induced PC synthesis correlates with a moderate increase of expression of genes involved in GSH synthesis, the change for gamma-ECS being most pronounced.

Amino Acid Sequence↗
PubMed2001

Substrate-specific regulation of the ribosome- translocon junction by N-terminal signal sequences.

Amino-terminal signal sequences target nascent secretory and membrane proteins to the endoplasmic reticulum for translocation. Subsequent interactions between the signal sequence and components of the translocation machinery at the endoplasmic reticulum are thought to be important for the productive engagement of the translocon by the ribosome-nascent chain complex. However, it is not clear whether all signal sequences carry out these posttargeting steps identically, or if there are differences in the interactions directed by one signal sequence versus another. In this study, we find substantial differences in the ability of signal sequences from different substrates to mediate closure of the ribosome--translocon junction early in translocation. We also show that these differences in some cases necessitate functional coordination between the signal sequence and mature domain for faithful translocation. Accordingly, the translocation of some proteins is sensitive to replacement of their signal sequences. In a particularly dramatic example, the topology of the prion protein was found to depend highly on the choice of signal sequence used to direct its translocation. Taken together, our results reveal an unanticipated degree of substrate-specific functionality encoded in N-terminal signal sequences.

Animals↗
PubMed1996

Identification of an additional member of the cytochrome c oxidase subunit VIIa family of proteins.

We report the cloning, nucleotide sequence, evolutionary analysis, and intracellular localization of SIG81, a silica-induced cDNA from mouse macrophages. The cDNA encodes a 111-amino acid protein with extensive sequence identity with members of the mammalian cytochrome c oxidase subunit VIIa (COX7a) family. A human SIG81 sequence >80% identical with the mouse cDNA was deducted from homologous sequences in the human expressed tags data base. The deduced aminoterminal region shows features common to mitochondrial targeting sequences. A phylogenetic analysis of the carboxyl-terminal domain homologous to COX7a identifies SIG81 as a divergent member of the family with an ancient origin. Southern blot analysis showed that the mouse genome contains two to three copies of the SIG81 gene. Northern blot analysis revealed that the SIG81 transcript is approximately 1 kb and expressed in every tissue tested, with higher levels of expression observed in kidney and liver. Antibodies raised against a glutathione S-transferase SIG81 fusion protein detected a 13.5-kDa protein that co-fractionates with mitochondrial localized enzymatic activity. Taken together, our data suggest that SIG81 is a novel member of the COX7a family that is constitutively expressed in mouse cells.

Amino Acid Sequence↗
PubMed2006

MEF immortalization to investigate the ins and outs of mutagenesis.

The importance of tumor suppressor/oncogene mutations in tumor development is clear, but the causes of the DNA sequence changes in human cancers are not. Although elegant experiments with transgenic mice harboring lacZ or cII target sequences show that exposure to mutagenic human carcinogens can cause base substitutions in vivo, it does not follow from this that the mutations found in human cancers have to be the direct result of damage by external mutagens. They could be due to endogenously generated reactive oxygen species, or polymerase infidelity, for example. Specific patterns of mutations in the defined sequence of a test system set up to address this question can provide information on the molecular events leading to DNA sequence changes in humans if the experimentally induced mutations and patient tumor mutations are compared in the same gene. Fortuitously, inactivating point mutations in the p53 gene are driving events in the immortalization of murine embryonic fibroblasts (MEFs) in vitro. This discovery offers a natural biological strategy for selecting p53 mutants. Immortalized cell lines arising from primary MEFs harboring human p53 sequences (Hupki, human p53 knock-in) have p53 mutations that match p53 mutations in human tumors.

Animals↗
PubMed1989

Purification of DNA-binding transcription factors by their selective adsorption on the affinity latex particles.

A simple method with the use of affinity latex particles has been developed for the fast and efficient purification of sequence-specific DNA-binding proteins on the basis of their ability to selectively bind to their target sequences. Complementary oligodeoxynucleotides that contained a recognition site for a sequence-specific DNA-binding protein were chemically synthesized, annealed and ligated to give oligomers. The oligomers were coupled to latex particles, composed of polyglycidyl methacrylate, using cyanogen bromide to yield affinity latex particles. The concentration of covalently bound DNA on the affinity latex particles was 6 times as much DNA per ml as that in the Sepharose resin conventionally used. By sequential batch-wise procedures with the affinity particles, one of the sequence-specific DNA binding transcription factors, ATF or E4TF3, was quickly and efficiently purified to homogeneity from either a protein fraction in which the factor was enriched or a crude cell extract.

Activating Transcription Factors↗
PubMed1998

Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines.

The synthesis of 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2'-deoxyadenosines is described. The nucleosides were synthesized, functionalized into the phosphoramidites and incorporated into oligodeoxynucleotides. Spectroscopic melting experiments against complementary RNA showed increases of 3-4 degreesC per modification for single substitutions and smaller increases per incorporation for multiple substitutions relative to unmodified control sequences. The 7-propyne and 7-iodo nucleosides were incorporated into antisense sequences targeting the 3'-UTR of murine C- raf mRNA. Both nucleosides demonstrated substitution-dependent potency. The sequences with three and four substitutions of the 7-propyne-7-deaza-2-amino-2'-deoxyadenosine exhibited a 2-3-fold increase in potency over unmodifed controls.

Animals↗
PubMed1987

L1 repeat elements in the human epsilon-G gamma-globin gene intergenic region: sequence analysis and concerted evolution within this family.

We have deduced the sequence of a composite long interspersed repeated DNA in primates and herein describe its relationship to a complex repeat element (L1Heg) located in the interval linking the human epsilon- and G gamma-globin genes. The main element of L1Heg is 3' truncated and interrupted by the insertion of the 3' end of a second L1 element. Transposition of L1Heg into this intergenic locus generated a 62-bp duplication of flanking sequences. In contrast, insertion of the second repeat may have been mediated by homology between donor and target sequences. The main repeat represents a novel class of abundant elements whose sequences have diverged from other rodent and primate LINES approximately 1.3 kb downstream from the 5' terminus of L1Heg. Comparison of L1Heg with the sequences of two other related L1 members revealed a complex set of rearrangements confined within a region that resembles the long terminal repeats of other types of retroposons. The boundaries of conversion-like events were defined on the basis of the clustering of nucleotide sequence variants common to two or more nonallelic 3' L1H elements. Several of these events are apparently initiated or resolved within a common 150-bp region that coincides with the 3' terminus of a pan-mammalian open reading frame. This analysis showed that concerted genetic interactions and random drift both contribute appreciably to sequence variation within this set of L1H members.

Animals↗
PubMed1991

Reverse-transcriptase-associated RNaseH activity mediates template switching during reverse transcription in vitro.

During the first steps of reverse transcription of the retroviral genome, sequences present at the extremities of the RNA are used to reconstitute a host cell PolII promoter. The assembly of the promoter occurs by template switching, which takes advantage of a direct repeat at the ends of the RNA molecule. These steps are catalysed by the viral reverse transcriptase, which carries an intrinsic RNaseH activity that is probably also involved therein. To study the role of the RNaseH activity in this first template-switching event, an in vitro system has been developed based on primer extensions of synthetic RNAs. When an RNA was reverse transcribed with wild-type reverse transcriptase in the presence of a second RNA the 3' part of which was repeated at the 5' end of the first one, extension products could be observed corresponding to a chimeric cDNA comprising both RNA species. This template switching could not be detected when a mutant reverse transcriptase lacking the RNaseH activity was used. The results show that the RNaseH activity is needed to remove the 5' RNA sequences from the cDNA:RNA hybrid thereby enabling its translocation to another RNA containing an appropriate complementary target sequence.

Endoribonucleases↗
PubMed1991

Illegitimate integration of non-replicative vectors in the genome of Rhodococcus fascians upon electrotransformation as an insertional mutagenesis system.

Electrotransformation of Rhodococcus fascians by non-replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences. Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.

Base Sequence↗