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PubMed1998

Improved polymerase chain reaction detection of clonally rearranged T-cell receptor beta chain genes.

A new method for the detection of all known possible rearrangements at the variable (V), diversity (D), and joining (J) segments of the T-cell receptor beta chain (TcR beta) gene in tissue DNA extracts is described that involves two polymerase chain reactions (PCRs). The first PCR round (screening PCR) allowed the identification of the J beta segment involved in a clonal rearrangement. A J beta-primer was used for the second PCR (J beta-specific PCR), recognizing the J beta segment identified in the screening PCR in combination with a consensus V beta primer. This PCR generated prominent and short amplificates suitable for direct sequence analysis because of their low background. Using this approach, clonal TcR beta gene rearrangements were able to be demonstrated in all T-cell lines (n = 7) and in all peripheral T-cell lymphomas (n = 33) analyzed. No clonal TcR beta gene rearrangements were found in any of the normal tissues studied nor in any B-cell non-Hodgkin lymphomas. This method is applicable to DNA from fresh frozen tissues, and, after the TcR beta rearrangement of a patient's malignant T-cell clone has been identified by the screening PCR, DNA can also be detected in follow-up formalin-fixed paraffin-embedded samples by the J beta-specific PCR with high sensitivity and specificity.

Clone Cells↗

PubMed1997

The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis.

The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a regulon, in which transcription of all three genes is activated in trans by the same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not observed in atx4-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes. First, total protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air were examined by two-dimensional electrophoresis. Comparison of the resulting protein patterns indicated that synthesis of non-toxin proteins is influenced by growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. Transposon-insertion libraries were screened for mutants expressing CO2-enhanced atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin coregulated) were Tox+, indicating that these genes may not be involved in anthrax toxin gene activation. Our data indicate a clear association of atxA with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA regulates genes other than the structural genes for the anthrax toxin proteins.

Antigens, Bacterial↗

PubMed1983

Transformation by iontophoretic microinjection of DNA: multiple integrations without tandem insertions.

DNA transformations of mouse tissue culture cells and mouse embryos were carried out by iontophoretic microinjection of DNA. Iontophoresis involves the use of an externally applied electric current to expel DNA molecules from the injection micropipette into the impaled cell (microelectrophoresis). Restriction analysis of transformants obtained by using this procedure demonstrated that this method gave strikingly different results from those previously obtained by others with transformants generated by pressure injection. First, tandem insertions were never observed in any of our transformants. Second, despite the lack of tandem insertions, we have obtained transformants which have integrated many copies of the injected DNA sequences, probably via a comparably large number of independent integration events. And third, in one transformant, an injected BamHI restriction fragment was found to have been integrated in its entirety; this included the preservation of the terminal BamHI recognition sequence. Based on these observations, we discuss the potential usefulness of iontophoretic DNA microinjections in DNA transformation studies that are focused either on the analysis of the regulation of gene expression or on the targeting of DNA sequences into the eucaryotic cell genome.

Animals↗

PubMed1992

Sampling methods in behavior research.

Animals perform a continuous stream of behavior throughout their lives. Because their behavior is not random, appropriate sampling methods can be used to obtain data that accurately reflect the actual behavior and are valid for answering research questions. Answering questions related to several variables assists in narrowing the choices of sampling methods. First, a determination must be made of what behaviors to measure. If the behaviors are few and easily measured, then All Occurrences Sampling is the method of choice because it generates accurate frequency and duration data through continuous recording. Sequence and Sociometric Matrix Sampling are specialized types of All Occurrences Sampling that are restricted to sampling intra- or interindividual sequences and social interactions (e.g., agonistic), respectively. Second, if who (e.g., specific individual, sex, or genotype) performs the behavior is a major component of the research question, then consideration should be given to Focal Animal (Pair, Group) Sampling. Third, if when or where the behavior is performed is of interest (e.g., activity budget), then Instantaneous or Scan Sampling can often be effective. Ad libitum Sampling does not produce valid data for analyses, but it is useful when formulating and fine-tuning research questions. One-Zero Sampling is not recommended except when the research question relates to the presence or absence of behaviors only. Other factors to consider in selecting a sampling method are duration of the behavior (event or state), desired scale of measurement (nominal, ordinal, interval, or ratio), and logistics (e.g., time, and equipment and facilities available).

Animals↗

PubMed1997

Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV.

Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Ambler's class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

Amino Acid Sequence↗

PubMed2000

Single-step conversion of P1 and P1 artificial chromosome clones into yeast artificial chromosomes.

Large insert genomic clones are useful for generating transgenic animals, particularly when specific mutations are introduced. To facilitate manipulation of large genomic sequences, we developed a method of converting Escherichia coli P1 artificial chromosomes (PACs) into yeast artificial chromosomes (YACs). A shuttle vector, pMAX-121, was generated that contains elements needed to generate a YAC (cen4, ars, ura3, his, and two telomere segments) along with approximately 1.3 kb of sequence homologous to P1 and PAC vector sequences. Cotransformation of yeast with the target PAC or P1 clone and pMAX-121 results in two homologous recombination events. The first, between the target clone and pMAX-121, results in a circular molecule. The second is an intramolecular recombination event between the two pMAX-121 telomere sequences, resulting in a linear molecule. The resulting YAC is stably maintained in yeast and can be further modified using homologous recombination. The method was used to convert a 201-kb PAC containing the human tau gene into a stable linear YAC. A second vector, pLys2-neo, was developed to retrofit the YAC with the yeast lys2 gene, a selectable marker replacing the yeast ura3 gene, and a Pgk-neo cassette that confers G418 resistance to mammalian cells. The resulting YAC can be used for generating transgenic animals and stably transfected cell lines. Also, the lys2 marker facilitates introduction of mutations by homologous recombination.

Bacteriophage P1↗

PubMed2002

Smoothelin contains a novel actin cytoskeleton localization sequence with similarity to troponin T.

Smoothelin, a cytoskeletal protein, exists in a large isoform specifically expressed in vascular smooth muscle cells, and a small visceral isoform generated by a downstream transcriptional start site. Using fusions to the green fluorescent protein, we could show that both smoothelin isoforms are localized at actin containing filaments and mapped two domains that are each sufficient for localization at the actin cytoskeleton. The first domain is located in the vascular-specific, N-terminal half of smoothelin and the second in the common, C-terminal half. The second domain shares clear sequence similarity with a domain of troponin T involved in actin filament association. These results suggest that the tissue-specific expression of smoothelin isoforms might contribute to the different contractile properties of smooth muscle cells.

Actins↗

PubMed1992

First and second moment of counts of words in random texts generated by Markov chains.

An exact expression for the variance of random frequency that a given word has in text generated by a Markov chain is presented. The result is applied to periodic Markov chains, which describe the protein-coding DNA sequences better than simple Markov chains. A new solution to the problem of word overlap is proposed. It was found that the expected frequency and overlapping properties determine most of the variance. The expectation and variance of counts for triplets are compared with experimental counts in Escherichia coli coding sequences.

Algorithms↗

PubMed2004

Generation of the BfiI restriction endonuclease from the fusion of a DNA recognition domain to a non-specific nuclease from the phospholipase D superfamily.

The BfiI endonuclease cleaves DNA at fixed positions downstream of an asymmetric sequence. Unlike other restriction enzymes, it functions without metal ions. The N-terminal half of BfiI is similar to Nuc, an EDTA-resistant nuclease from Salmonella typhimurium that belongs to the phosphoplipase D superfamily. Nuc is a dimer with one active site at its subunit interface, as is BfiI, but it cuts DNA non-specifically. BfiI was cleaved by thermolysin into an N-terminal domain, which forms a dimer with non-specific nuclease activity, and a C-terminal domain, which lacks catalytic activity but binds specifically to the recognition sequence as a monomer. On denaturation with guanidinium, BfiI underwent two unfolding transitions: one at a relatively low concentration of guanidinium, to a dimeric non-specific nuclease; a second at a higher concentration, to an inactive monomer. The isolated C-terminal domain unfolded at the first (relatively low) concentration, the isolated N-terminal at the second. Hence, BfiI consists of two physically separate domains, with catalytic and dimerisation functions in the N terminus and DNA recognition functions in the C terminus. It is the first example of a restriction enzyme generated by the evolutionary fusion of a DNA recognition domain to a phosphodiesterase from the phospholipase D superfamily. BfiI may consist of three structural units: a stable central core with the active site, made from two copies of the N-terminal domain, flanked by relatively unstable C-terminal domains, that each bind a copy of the recognition sequence.

Bacterial Proteins↗

PubMed1993

Does Escherichia coli possess a second citrate synthase gene?

Escherichia coli possesses a hexameric citrate synthase that exhibits allosteric kinetics and regulatory sensitivity, and for which the gene (gltA) has previously been cloned and sequenced. A citrate-synthase-deficient strain of E. coli (K114) has been mutated to generate a revertant (K114r4) that produces a dimeric citrate synthase with altered kinetic and regulatory properties. On cloning and sequencing the gltA gene from both K114 and K114r4, a single mutation was found that caused the replacement of Asp362 with Asn. Asp362 has been previously shown to be a catalytically essential residue in E. coli citrate synthase, and we demonstrate that the hexameric enzyme produced on expression of the gltA gene from K114 and K114r4 is inactive. The dimeric citrate synthase from K114r4 has been purified and shown to be immunologically distinct from the wild-type hexameric enzyme. Determination of its N-terminal amino acid sequence demonstrates that the mutant citrate synthase is encoded by a gene distinct from the E. coli gltA gene. The N-terminal sequence is compared with those of other eukaryotic, eubacterial and archaebacterial citrate synthases.

Amino Acid Sequence↗

PubMed2002

On the roles of repetitive DNA elements in the context of a unified genomic-epigenetic system.

Repetitive DNA sequences comprise a substantial portion of most eukaryotic and some prokaryotic chromosomes. Despite nearly forty years of research, the functions of various sequence families as a whole and their monomer units remain largely unknown. The inability to map specific functional roles onto many repetitive DNA elements (REs), coupled with the taxon-specificity of sequence families, have led many to speculate that these genomic components are "selfish" replicators generating genomic "junk." The purpose of this paper is to critically examine the selfishness, evolutionary effects, and functionality of REs. First, a brief overview of the range of ideas pertaining to RE function is presented. Second, the argument is presented that the selfish DNA "hypothesis" is actually a narrative scheme, that it serves to protect neo-Darwinian assumptions from criticism, and that this story is untestable and therefore not a hypothesis. Third, attempts to synthesize the selfish DNA concept with complex systems models of the genome and RE functionality are critiqued. Fourth, the supposed connection between RE-induced mutations and macroevolutionary events are stated to be at variance with empirical evidence and theoretical considerations. Hypotheses that base phylogenetic transitions in repetitive sequence changes thus remain speculative. Fifth and finally, the case is made for viewing REs as integrally functional components of chromosomes, genomes, and cells. It is argued throughout that a new conceptual framework is needed for understanding the roles of repetitive DNA in genomic/epigenetic systems, and that neo-Darwinian "narratives" have been the primary obstacle to elucidating the effects of these enigmatic components of chromosomes.

Animals↗

PubMed1982

Sequence-specific cleavage of chromatin by staphylococcal nuclease can generate an atypical nucleosome pattern.

We have investigated the nucleosomal organization of ribosomal genes in the acellular slime mold Physarum polycephalum. When probed with staphylococcal nuclease, the ribosomal genes appear to be uniformly packed in nucleosomes, in an arrangement which is indistinguishable from the pattern obtained with bulk chromatin. During this study, an unusual pattern of digestion was obtained from a DNA region immediately upstream of the initiation site of rRNA transcription, in addition to the nucleosomal profile, a second regular ladder of fragments with a repeat length of 30-40 basepairs was generated from this region. We established that this pattern of degradation reflects the strong preference of staphylococcal nuclease for certain nucleotide arrangements on the DNA, rather than a particular chromatin configuration. These observations clearly show that great caution needs to be exerted whenever data from staphylococcal nuclease digestions are interpreted in terms of chromatin structure.

Base Sequence↗

PubMed1979

Some sequence similarities among cloned mouse DNA segments that code for lambda and kappa light chains of immunoglobulins.

A comparison between the cloned mouse DNA segments that were found to code for the lambda and kappa light chains of immunoglobulins established that there were seven short nucleotide sequences, two of which matched 6 out of 7, two 7 out of 8, two 8 out of 9, and one 9 out of 10 bases; these sequences were located either at homologous amino acid positions or at positions displaced by four amino acids or less. They all occurred in the framework regions (FRs), five next to the complementarity-determining regions (CDRs). Three of these were unique and did not occur elsewhere in the immunoglobulin nucleotides sequenced thus far or in DNA's of phage phi X174, phage G4, or simian virus 40. Five could serve as sites of joining by recombination or insertion of CDR to FR segments, and the invariant tryptophan that is the first residue of the second FR might serve as a sixth. These sites are consistent with the mini-gene or insertional hypotheses for the generation of antibody diversity but could also serve as points of recognition for a mutator enzyme or could serve to limit somatic mutation to the CDRs.

Amino Acid Sequence↗

PubMedDate not supplied

Intracellular glycosylation and active efflux as mechanisms for resistance to oleandomycin in Streptomyces antibioticus, the producer organism.

Resistance to macrolides in producing organisms can be achieved by target site modification, intracellular inactivation of the antibiotic or active efflux mechanisms for the excretion of the antibiotic. The oleandomycin producer, Streptomyces antibioticus, possesses oleandomycin-sensitive ribosomes all along the cell cycle. However, it contains an intracellular glycosyltransferase capable of inactivating oleandomycin in the presence of UDP-glucose as cofactor. The correspondent gene (oleD) has been cloned and sequenced and the glycosyltransferase purified. Two other genes (oleB and oleC) that confer oleandomycin resistance have been cloned and characterized and both encode ABC (ATP-Binding Cassette) transporters. These may constitute the excretion mechanism throughout which the glycosylated oleandomycin is excreted. A second enzyme activity has been purified from culture supernatants of the oleandomycin producer that releases the glucose from the inactive glycosylated oleandomycin generating active antibiotic. This enzyme would probably catalyse the last step in the biosynthesis of oleandomycin.

Amino Acid Sequence↗

PubMed1994

Alternate-strand DNA triple-helix formation using short acridine-linked oligonucleotides.

We have used DNAse I footprinting to examine the formation of intermolecular DNA triple helices at sequences containing adjacent blocks of purines and pyrimidines. The target sites G6T6.A6C6 and T6G6.C6A6 were cloned into longer DNA fragments and used as substrates for DNAse I footprinting, which examined the binding of the acridine (Acr)-linked oligonucleotides Acr-T5G5 and Acr-G5T5 respectively. These third strands were designed to incorporate both G.GC triplets, with antiparallel Gn strands held together by reverse Hoogsteen base pairs, and T.AT triplets, with the two T-containing strands arranged antiparallel to each other. We find that Acr-T5G5 binds to the target sequence G6T6.-A6C6, in the presence of magnesium at pH 7.0, generating clear DNAse I footprints. In this structure the central guanine is not recognized by the third strand and is accessible to modification by dimethyl sulphate. Under these conditions no footprint was observed with Acr-G5T5 and T6G6.C6A6, though this triplex was evident in the presence of manganese chloride. Manganese also facilitated the binding of Acr-T5G5 to a second site in the fragment containing the sequence T6G6.C6A6. This represents interaction with the sequence G4ATCT6, located at the boundary between the synthetic insert and the remainder of the fragment, and suggests that this bivalent metal ion may stabilize triplexes that contain one or two mismatches. Manganese did not affect the interaction of either oligonucleotide with G6T6.A6C6.

Acridines↗

PubMedDate not supplied

[Fundamental aspects of molecular biology. The multigene family of 5-HT1 receptors].

Genes, constituted by the deoxyribonucleic acid (DNA) contain all the genetic information of a cell. DNA is a double helix consisting of two antiparallel polynucleotide chains where each nucleotide is composed of the association between a base, a sugar and a phosphate group. The chains are associated by hydrogen bonds between the bases. Guanine bonds specifically with cytosine while adenine bonds specifically with thymine. The sequence of DNA is related to the sequence of protein by the genetic code. Each aminoacid is represented by a codon that consists of a nucleotide triplet. Information is expressed by a two-step process. The first step, transcription generates a single-stranded ribonucleic acid called messenger RNA (mRNA) (where a uracil base is present instead of a thymine) which acts as an intermediate molecule. In this step, RNA splicing is one of the maturation processes consisting of joining the exons by removing the introns of the precursor RNA molecule. The second step, translation, converts the nucleotide sequence into the sequence of aminoacids. Since the 1970s, the progress in DNA technology consisting of the development of molecular cloning, DNA library construction and sequencing methods, has made it possible to isolate and analyze specific genes directly from the genome. A sequence of genomic or complementary DNA (cDNA) is cloned by inserting it into a vector (plasmid or phage) that can replicate independently in bacteria. Before cloning, genomic DNA is digested into fragments by restriction enzymes (endonucleases cleaving specific sequences within double-stranded DNA) whereas RNA sequences are copied into cDNA by reverse transcriptase.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

The rat P450IIE1 gene: complete intron and exon sequence, chromosome mapping, and correlation of developmental expression with specific 5' cytosine demethylation.

The gene coding for the ethanol-inducible, developmentally regulated P450IIE1 was isolated from an lambda EMBL 3 rat genomic library and completely sequenced. The gene spanned 10,373 base pairs and contained nine exons. Upstream and downstream DNA of 1530 and 825 base pairs, respectively, was also sequenced, and the transcription start site was identified by both S1 mapping and primer extension. A typical TATA box was found just upstream of the start site; however, no CCAAT box was apparent. Other repetitive sequences were identified including a partial R.dre.1 sequence upstream of the gene and a long stretch of 160 alternating purines in the second intron. This latter repetitive element is found in many mammalian genes. By use of a panel of mouse-hamster somatic cell hybrids, the P450IIE1 gene was localized to mouse chromosome 7. The hepatic P450IIE1 gene is transcriptionally activated within 1 day after birth and reaches a maximal level of expression at 6 days of age. Using restriction endonuclease sites generated from the gene sequence data and the cytosine methylation-sensitive enzymes HhaI and HpaII, we found that this transcriptional activation during early development is coincident with specific demethylation only at the 5' end of the P450IIE1 gene. Interestingly, other cytosine residues in the middle of the gene became demethylated as rats aged from 1 to 10 weeks, at which time no changes in gene expression occur.

Amino Acid Sequence↗

PubMed1990

Structure and polymorphic map of human lipoprotein lipase gene.

Lipoprotein lipase (LPL) catalyzes the key step for the removal of triacylglycerol-rich lipoproteins from the circulation. In this paper, we report the cloning and structure of the normal human LPL gene, which was isolated in three overlapping lambda phage clones that span about 35 kilo bases (kb) of the genetic locus. The peptide coding region of the gene is approx. 23 kb in length and contains nine exons with intron sizes ranging from 0.7 to 8.7 kb. The entire 3' untranslated region is in the tenth exon. Specific sequences in this region support the hypothesis that two mRNA species found for human LPL are generated by differential utilization of polyadenylation signals. The first exon occurs in the 5' untranslated region and the region coding for the signal peptide. The second exon includes the protein domain coding for the N-linked glycosylation site that is required for the expression of enzyme activity. The fourth exon contains the region that was proposed as a lipid binding domain, the sixth for one putative heparin binding domain, and the eighth codes for a domain containing another N-linked glycosylation site. These results suggest that the unique structural and functional domains are confined to specific exons. The PvuII polymorphic site was located within the intron between exon 6 and 7 and the HindIII polymorphic site to the 3' flanking region. The location of these polymorphic sites suggests that the PvuII restriction fragment length polymorphism (RFLP) associated with lipase deficiency in a few Japanese kindred may be a linkage marker for a functional defect of LPL, while the HindIII RFLP associated with hypertriglyceridemia may be important for gene regulation of LPL.

Amino Acid Sequence↗