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PubMed1994

Nucleotide sequence analysis of enteropathogenic Escherichia coli (EPEC) adherence factor probe and development of PCR for rapid detection of EPEC harboring virulence plasmids.

The 1-kb BamHI-SalI fragment from plasmid pMAR2 termed the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) probe was cloned in pUC19 and pK18. The nucleotide sequence of this fragment was determined, and a set of primers was designed to amplify a 397-bp region associated with pMAR2 by PCR. An analysis of the whole EAF sequence with database libraries indicated no significant homology to any known genes. However, between bases 701 and 787 of the fragment, an 82.8% homology between the EAF and the insertion sequence IS630 of Shigella sonnei exists. The results of PCR with primers of the EAF sequence demonstrated that all of the 151 EAF probe-positive EPEC strains with localized adherence to HEp-2 cells yielded positive EAF PCR results. In contrast, none of the 277 EAF probe-negative strains reacted to the EAF PCR. In addition, the PCR assay was successfully used to generate vector-free digoxigenin-labeled EAF fragments that gave valid results in colony blot hybridization assays. The EAF PCR appears to be a specific and efficient method for the detection of EPEC strains carrying the EAF plasmids.

Adhesins, Escherichia coli↗

PubMed1996

The central Z-disk region of titin is assembled from a novel repeat in variable copy numbers.

The giant sarcomeric protein titin (also described as connectin) is composed mainly of immunoglobulin (Ig)-like and fibronectin type III (fn3)-like domains arranged consecutively. At both ends of the molecule, these domains are interrupted by sequence insertions. The amino terminus of titin is localized in the Z-disk, a structure of great variability in different muscle types. We have determined the ultrastructural position of sequences in this region of the molecule in skeletal and cardiac muscle by immunoelectron microscopy using antibodies directed against unique epitopes. Titin molecules entering the Z-disk from two half sarcomeres do not significantly overlap, showing that the amino terminus is at the centre of the Z-disk. A serine/proline rich site, which can be phosphorylated by kinases in developing muscle tissues, was identified near the amino terminus of titin. Sequence analysis revealed the presence of a novel 45 residue repeat ('Z-repeats') in this region of the molecule. The number of titin Z-repeats varies due to differential splicing. We propose that this mechanism is a means of assembling Z-disks of variable thickness and mechanical strength.

Amino Acid Sequence↗

PubMed1980

Comparison of Epstein-Barr virus strains of different origin by analysis of the viral DNAs.

Epstein-Barr virus (EBV) originating from Burkitt's lymphoma (P3HR-1 and CC34-5), nasopharyngeal carcinoma (M-ABA), transfusion mononucleosis (B95-8), and a patient with acute myeloblastic leukemia (QIMR-WIL) was isolated from virus-carrying lymphoid cell lines after induction with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate. Viral DNA was analyzed by partial denaturation mapping and by use of the restriction endonucleases EcoRI, HindIII, and SalI and separation of fragments in 0.4% agarose. By using the restriction enzyme data of B95-8 (EBV) and W91 (EBV) obtained by Given and Kieff (D. Given and E. Kieff, J. Virol. 28:524-542, 1978), maps were established for the other virus strains. Comigrating fragments were assumed to be identical or closely related among the different strains. Fragments of different strains migrating differently were isolated, purified, radioactively labeled, and mapped by hybridization against blots of separated viral fragments. The results were as follows. (i) All strains studied were closely related. (ii) The number of internal repeats was variable among and within viral strains. (iii) B95-8 (EBV) was the only strain with a large deletion of about 12,000 base pairs at the right-hand side of the molecule. At the same site, small deletions of about 400 to 500 base pairs were observed in P3HR-1 (EBV) and M-ABA (EBV) DNA. (iv) P3HR-1 (EBV), the only nontransforming EBV strain, had a deletion of about 3,000 to 4,000 base pairs in the long unique region adjacent to the internal repeats carrying a HindIII site. (v) Small inserted sequences of 150 to 400 base pairs were observed in M-ABA (EBV) and B95-8 (EBV) at identical sites in the middle of the long unique region. (vi) Near this site, an insertion of about 1,000 base pairs was found in P3HR-1 (EBV) DNA. (vii) The cleavage patterns of P3HR-1 virus DNA and the results of blot hybridizations with P3HR-1 virus fragments are not conclusive and point to the possibility that in addition to the normal cleavage pattern some viral sequences may be arranged differently. Even though it is possible that small differences in the genome organization may have significant biological effects, the great similarity among different EBV strains does not favor the hypothesis that disease-specific subtypes exist.

Base Sequence↗

PubMed2002

Selenoproteins and selenocysteine insertion system in the model plant cell system, Chlamydomonas reinhardtii.

Known eukaryotic selenocysteine (Sec)-containing proteins are animal proteins, whereas selenoproteins have not been found in yeast and plants. Surprisingly, we detected selenoproteins in a member of the plant kingdom, Chlamydomonas reinhardtii, and directly identified two of them as phospholipid hydroperoxide glutathione peroxidase and selenoprotein W homologs. Moreover, a selenocysteyl-tRNA was isolated that recognized specifically the Sec codon UGA. Subsequent gene cloning and bioinformatics analyses identified eight additional selenoproteins, including methionine-S-sulfoxide reductase, a selenoprotein specific to Chlamydomonas: Chlamydomonas selenoprotein genes contained selenocysteine insertion sequence (SECIS) elements that were similar, but not identical, to those of animals. These SECIS elements could direct selenoprotein synthesis in mammalian cells, indicating a common origin of plant and animal Sec insertion systems. We found that selenium is required for optimal growth of Chlamydomonas: Finally, evolutionary analyses suggested that selenoproteins present in Chlamydomonas and animals evolved early, and were independently lost in land plants, yeast and some animals.

Amino Acid Sequence↗

PubMed2006

An inserted loop region of stromal ascorbate peroxidase is involved in its hydrogen peroxide-mediated inactivation.

Ascorbate peroxidase isoforms localized in the stroma and thylakoid of higher plant chloroplasts are rapidly inactivated by hydrogen peroxide if the second substrate, ascorbate, is depleted. However, cytosolic and microbody-localized isoforms from higher plants as well as ascorbate peroxidase B, an ascorbate peroxidase of a red alga Galdieria partita, are relatively tolerant. We constructed various chimeric ascorbate peroxidases in which regions of ascorbate peroxidase B, from sites internal to the C-terminal end, were exchanged with corresponding regions of the stromal ascorbate peroxidase of spinach. Analysis of these showed that a region between residues 245 and 287 was involved in the inactivation by hydrogen peroxide. A 16-residue amino acid sequence (249-264) found in this region of the stromal ascorbate peroxidase was not found in other ascorbate peroxidase isoforms. A chimeric ascorbate peroxidase B with this sequence inserted was inactivated by hydrogen peroxide within a few minutes. The sequence forms a loop that binds noncovalently to heme in cytosolic ascorbate peroxidase of pea but does not bind to it in stromal ascorbate peroxidase of tobacco, and binds to cations in both ascorbate peroxidases. The higher susceptibility of the stromal ascorbate peroxidase may be due to a distorted interaction of the loop with the cation and/or the heme.

Amino Acid Sequence↗

PubMed2004

Analysis of the N-terminal DNA binding domain of the IS30 transposase.

IS30 is the founding member of a large family of widely spread bacterial insertion sequences with closely related transposases. The N-terminal end of the IS30 transposase had been shown to retain sequence-specific DNA binding activity and to protect the IS30 terminal inverted repeats. Structural predictions revealed the presence of a helix-helix-turn-helix motif (H-HTH2) which, in the case of IS30, is preceded by an additional helix-turn-helix motif (HTH1). Analysis of deletion and point mutants in this region revealed that both motifs are important for IS30 transposition. IS30 exhibits two types of insertion specificity preferring either a 24 bp palindromic hot-spot (GOHS) or sequences resembling its ends [left and right terminal inverted repeat (IRL and IRR)]. Results are presented suggesting that the HTH1 region is required for GOHS targeting and interferes with the inverted repeat (IR) targeting. On the other hand, H-HTH2 appears to be required for both. The binding activities of the mutant proteins to the terminal IS30 IRs as measured by gel retardation correlated well with these results. Furthermore, close inspection of the H-HTH2 region revealed significant amino acid identity with a similar predicted secondary structure carried by the transcriptional regulator FixJ of Sinorhizobium meliloti and involved in FixJ binding to its target sequence. This suggests that FixJ and IS30 transposase share similar sequence-specific DNA binding mechanisms.

Amino Acid Sequence↗

PubMed1990

Regions of the Moloney murine leukemia virus genome specifically related to induction of promonocytic tumors.

Moloney murine leukemia virus (MuLV) can be a potent inducer of promonocytic leukemias in mice that are undergoing a chronic inflammatory response. The neoplasms are, at least in part, associated with insertional mutagenesis of the c-myb locus. Evidence is presented for the existence of at least two genetic elements of the virus that are crucial to induction of this disease but are not required for viral replication in hematopoietic tissues or induction of lymphoid disease. These genetic elements were detected by testing the pathogenicity of recombinants between Moloney and Friend MuLVs, the latter of which is nonleukemic to myeloid cells under these conditions, and by testing Moloney MuLV-based viruses that have nonretroviral sequences inserted at specific endonuclease sites in their long terminal repeats (LTRs). Analysis of the Moloney/Friend recombinants showed that there are sequences within the structural gene domain of Moloney, but not Friend, MuLV that are necessary for promonocytic leukemia, whereas the LTRs of the MuLVs are equally effective for promonocytic tumor formation and insertional mutagenesis of the c-myb gene. Experiments with viruses which were mutagenized in the LTR by insertions demonstrated that there is a specific genetic element in the U3 region of the LTR of Moloney MuLV, upstream of the 75-base-pair enhancer which, when interrupted, results in loss of leukemogenicity for cells in the monocytic lineage but not cells in the lymphoid lineage. We conclude, therefore, that promonocytic leukemia induction, in Moloney MuLV-infected mice undergoing a chronic inflammatory response, requires specific sequences in the structural gene region of Moloney MuLV as well as other sequences in the regulatory region of the virus.

Animals↗

PubMed2001

Suppression subtractive hybridization for identifying differentially expressed genes in renal cell carcinoma.

OBJECTIVE: To construct a renal cell carcinoma (RCC) cDNA subtractive library using suppression subtractive hybridization. METHODS: Polyadenylated RNA [Poly (A)+ RNA] was isolated from tissues of RCC and normal kidney, and single-strand cDNAs and double-strand cDNAs were synthesized in turn. RCC cDNAs were divided into two groups and ligated to the specific adaptors I and 2, and then hybridized with normal kidney cDNA twice with two rounds of suppression PCR. Second round PCR products were cloned to T/A plasmid vectors to set up the subtractive library. One hundred clones were randomly picked to perform enzyme digest analysis, and some underwent sequence analysis and Northern blot to identify RCC specifically expressed genes. SMART RACE procedure was operated to clone full length novel RCC specifically expressed genes. RESULTS: A human RCC subtractive library with high subtractive efficiency was successfully set up. The amplified library contains 350 positive clones. Random analysis of 100 clones with enzyme restriction showed that 85 plasmids in the clones contained 50-400 bp inserts. Sequence analysis was performed for 10 clones. All the 10 sequences were unknown before and derived from 6 unique, novel genes among which the cDNA insert RCC18 had five copies. Northern blot analysis showed that RCC18 cDNA was highly expressed in RCC, but no signal could be detected in normal kidney. Using SMART RACE technique, we obtained the full length of the novel gene RCC18. CONCLUSIONS: The constructed cDNA subtractive library of human RCC is a highly efficient one and lays a solid foundation for large scale screening and cloning new and specific oncogenes or tumor suppressor genes of RCC. The novel specifically expressed genes provided an important clue for studying the mechanisms of occurrence and development of RCC.

Carcinoma, Renal Cell↗

PubMed1993

Identification of a putative infC-rpmI-rplT operon flanked by long inverted repeats in Mycoplasma fermentans (incognitus strain).

A specific 1542-bp DNA fragment was amplified from Mycoplasma fermentans (incognitus strain) using a unique 23-nucleotide (nt) synthetic deoxyribonucleotide (oligo) (5'-TCCAAAAAGTCCGGAATTTGGGG) as the primer pair in the polymerase chain reaction (PCR). The 23-nt sequence is part of the 29-bp terminal inverted repeat (IR) which forms the left potential stem-and-loop (s&l) structure of the previously identified M. fermentans insertion-sequence(IS)-like genetic element [Hu et al., Gene 93 (1990) 67-72]. The amplified DNA was cloned and sequenced. A pair of 27-bp IR containing the 23-nt synthetic oligo was identified at both termini. Between the IR, there are four potential open reading frames (ORFs) which are arranged adjacent to each other in the order, ORF-1, ORF-2, ORF-3 and ORF-4, with parts of ORF-1 and ORF-2 overlapping. The deduced amino acid (aa) sequences of ORF-2, ORF-3 and ORF-4 are 34 to 60% identical to the translation initiation factor IF3 (encoded by the infC gene), ribosomal proteins L35 (rpmI gene) and L20 (rplT gene) of Escherichia coli and Bacillus stearothermophilus, respectively. In bacteria, the infC-rpmI-rplT genes are organized to function as an operon. There are multiple sites with promoter-like sequences identified upstream from the putative infC gene in the mycoplasma closely resembling the gene arrangement in the bacterial operon. All three genes of ORF-2, ORF-3 and ORF-4 are preceded individually by a strong appropriately spaced (7 and 10 bp) putative Shine-Dalgarno sequence (5'-AAGGA).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed2000

Complete DNA sequence of a serogroup A strain of Neisseria meningitidis Z2491.

Neisseria meningitidis causes bacterial meningitis and is therefore responsible for considerable morbidity and mortality in both the developed and the developing world. Meningococci are opportunistic pathogens that colonize the nasopharynges and oropharynges of asymptomatic carriers. For reasons that are still mostly unknown, they occasionally gain access to the blood, and subsequently to the cerebrospinal fluid, to cause septicaemia and meningitis. N. meningitidis strains are divided into a number of serogroups on the basis of the immunochemistry of their capsular polysaccharides; serogroup A strains are responsible for major epidemics and pandemics of meningococcal disease, and therefore most of the morbidity and mortality associated with this disease. Here we have determined the complete genome sequence of a serogroup A strain of Neisseria meningitidis, Z2491. The sequence is 2,184,406 base pairs in length, with an overall G+C content of 51.8%, and contains 2,121 predicted coding sequences. The most notable feature of the genome is the presence of many hundreds of repetitive elements, ranging from short repeats, positioned either singly or in large multiple arrays, to insertion sequences and gene duplications of one kilobase or more. Many of these repeats appear to be involved in genome fluidity and antigenic variation in this important human pathogen.

Antigenic Variation↗

PubMed1999

Interspecies recombination between enterococci: genetic and phenotypic diversity of vancomycin-resistant transconjugants.

Handwerger and colleagues demonstrated that a particular clinical isolate of Enterococcus faecium, designated GUC, and here redesignated as GUCR, can conjugatively transfer vancomycin resistance. The vancomycin resistance is encoded by a chromosomally born linked set of genes in the donor, designated the vanA cluster, to the chromosome of an E. faecalis recipient, JH2-2. Here it is reported that an earlier isolate of E. faecium from the same patient who later harbored the vancomycin-resistant E. faecium GUCR lacks the vanA gene cluster but is otherwise similar (by SmaI chromosomal fingerprint and metabolic fingerprinting) to the vancomycin-resistant GUCR. Therefore, "GUCS" is a strong suspect as the base strain for the clinical acquisition of the vanA cluster present in GUCR. Thirteen laboratory-generated vanA transconjugants derived from conjugation between GUCR and JH2-2 were subjected to further analysis, allowing a comparison between transfer in the laboratory and transfer that occurred in the clinical setting. Surprisingly, each JH2-2 transconjugant had a unique constellation of abilities to oxidize various members of a panel of potential carbon sources. This pattern was stable for each transconjugant, and it was not changed by growing the strains with or without vancomycin. Transconjugants had pulsed-field gel electrophoretic (PFGE) patterns largely consistent with that of JH2-2, the recipient in conjugation experiments. However, PFGE analysis showed that a large but variable amount of DNA, between 145 kb and 277 kb, was transferred into different transconjugants. The mechanism appears to be conjugative transposition in which new DNA is added to the pre-existing genome rather than substituting for a segment in the recipient. Mapping and hybridization studies of several transconjugants showed that each received similar, but not exactly the same, DNA fragment of at least 30 kb from the donor. Sequencing of 16S ribosomal genes was used to confirm that the recipient and donor strains used in transconjugation experiments were different species. Sequence analysis was also used to consider the possibility that rRNA operons might be mobilized in conjugation, but no evidence for the transfer of rDNA operons was found. An apparent insertion sequence in E. faecium almost identical to IS 1485 and 57% sequence identity to IS 199 of Streptococcus mutans was found in the region of DNA transferred. The results imply new consequences of conjugative transfer and interspecies recombination.

Anti-Bacterial Agents↗

PubMed1983

Carp preproinsulin cDNA sequence and evolution of insulin genes.

The nucleic acid sequence of the preproinsulin cDNA of carp (Cyprinus carpio), cloned in the PstI-site of pBR322 (1), has been determined. The sequenced insert of 439 bp includes the complete coding information for carp preproinsulin (108 amino acids), 10 nucleotides of the 5'-and 105 nucleotides of the 3'-nontranslated regions. The nucleotide sequence confirms the previously established amino acid sequence of carp insulin (2) and determines those of the signal (21 aa 1) and C-peptide (35 aa 1). The observed shortness of the signal peptide of carp preproinsulin and the N-terminal addition of 2 amino acids to the carp insulin B-chain suggest that the cleavage site of the signal peptidase has moved. Calculations based on the comparison of known preproinsulin cDNA sequences showed that the evolutionary distance between fresh water and salt water teleostians is not smaller than between man and chicken.

Amino Acid Sequence↗

PubMed1992

A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins.

We have generated transgenic mice that express a diverse repertoire of human sequence immunoglobulins. The expression of this repertoire is directed by light and heavy chain minilocus transgenes comprised of human protein coding sequences in an unrearranged, germ-line configuration. In this paper we describe the construction of these miniloci and the composition of the CDR3 repertoire generated by the transgenic mice. The largest transgene discussed is a heavy chain minilocus that includes human mu and gamma 1 coding sequences together with their respective switch regions. It consists of a single 61 kb DNA fragment propagated in a bacterial plasmid vector. Both human heavy chain classes are expressed in animals that carry the transgene. In light chain transgenic animals the unrearranged minilocus sequences recombine to form VJ joints that use all five human J kappa segments, resulting in a diversity of human-like CDR3 regions. Similarly, in heavy chain transgenics the inserted sequences undergo VDJ joining complete with N region addition to generate a human-like VH CDR3 repertoire. All six human JH segments and at least eight of the ten transgene encoded human D segments are expressed. The transgenic animals described in this paper represent a potential source of human sequence antibodies for in vivo therapeutic applications.

Animals↗

PubMed2002

Pyrococcus genome comparison evidences chromosome shuffling-driven evolution.

The genomes of three Pyrococcus species, P.abyssi, P.furiosus and P.horikoshii, were compared at the DNA level, taking advantage of our identification of their replication origins. Three types of rearrangements have been identified: (i) inversion and translation across the replication axis (origin/terminus), (ii) inversion and translocation restricted to a replichore (the half chromosome divided by the replication axis) and (iii) apparent mobility of long clusters of repeated sequences. Rearrangements restricted within a replichore were more common between P.furiosus and the two other Pyrococcus species than between P.horikoshii and P.abyssi. A strong correlation was found between 23 homologous insertion sequence elements, present only in P.furiosus, and recombined segment boundaries, suggesting that transposition events have been a major cause of genomic disruption in this species. Moreover, gene orientation bias was much more disrupted than strand composition biases in fragments that switched their orientation within a replichore upon recombination. This allowed us to conclude that one reversion and one translation occurred in P.abyssi after its divergence from P.horikoshii, and that a smaller segment has specifically recombined in P.furiosus. Whereas a majority of genes are transcribed in the same direction as DNA replication in P.horikoshii and P.abyssi, the colinearity of transcription and replication is only maintained for highly transcribed genes in P.furiosus. We discuss the implications of genomic rearrangements on gene orientation and composition biases, and their consequences on sequence evolution.

Chromosome Inversion↗

PubMed1980

A bacterial cell that synthesizes a protein containing the antigenic determinants of rat prolactin.

Bacterial minicells containing three different recombinant plasmids with rat prolactin cDNA sequences inserted at the Pst I site of pBR322 via the poly(dG):poly(dC) joining technique were examined for the expression of rat prolactin antigenic determinants. The three prolactin coding sequences were in the same orientation as the coding sequence of the ampicillin-resistance gene of pBR322. The presence of each of the three recombinant plasmids induced some prolactin synthesis by the bacteria as measured by immunoprecipitation with anti-prolactin antisera. About 10% of the protein synthesized from one of the plasmids, prl 3, precipitated with the antisera. These prolactin antigenic determinants were part of a larger fused protein.

Animals↗

PubMed1984

Carp preproinsulin cDNA sequence and evolution of insulin genes.

The nucleic acid sequence of the preproinsulin cDNA of carp (Cyprinus carpio), cloned in the PstI site of pBR322 ( Liebscher et al. 1980), has been determined. The sequenced insert of 439 bp includes the complete coding information for carp preproinsulin (108 amino acids), 10 nucleotides of the 5'-and 105 nucleotides of the 3'-nontranslated regions. The nucleotide sequence confirms the previously established amino acid sequence of carp insulin ( Makower et al. 1982) and determines those of the signal 21 amino acids and C peptide (35 amino acids). The observed shortness of the signal peptide of carp preproinsulin and the N-terminal addition of 2 amino acids to the carp insulin B chain suggest that the cleavage site of the signal peptidase has moved. Calculations based on the comparison of known preproinsulin cDNA sequences showed that the evolutionary distance between fresh water and salt water teleostians is not smaller than that between man and chicken.

Amino Acid Sequence↗

PubMedDate not supplied

Creation of ultra-rare restriction sites in intact eucaryotic chromosomes mediated by bacterial methylases: an approach to sequencing and analyzing tumor and normal genomes.

The limited restriction of eucaryotic chromosomes would facilitate our understanding of the aberrant genomes of genetic diseases and cancer. We have described methods for methylating eucaryotic chromosomes embedded in agarose plugs. We now describe how Cla I methylase can be utilized in a methylation-dependent restriction cleavage with Dpn I to restrict eucaryotic genomes into a limited number of fragments. We have restricted the genomes of Saccharomyces cerevisiae at four sites and cleaved the three chromosomes of Schizosaccharomyces pombe into eleven fragments. Unlike the recently published methodologies using DNA sequences inserted into procaryotic and eucaryotic genomes, the methodologies described here use unaltered eucaryotic genomes for highly limited restriction. This methodology has applications in speeding, simplifying, and reducing the cost of sequencing the human genome.

Bacterial Proteins↗

PubMed1996

Expression of TrkB receptor isoforms in the developing avian visual system.

The expression of novel TrkB receptor transcripts has been characterized to understand the potentially diverse roles of brain-derived neurotrophic factor (BDNF) in the developing avian visual system. In situ localization with an extracellular domain probe common to all TrkB transcripts labeled a sub-population of large retinal ganglion cells as well as many associated visual nuclei, including the neuronal layers within the tectum that receive retinal innervation. Because of the potential for structurally and functionally distinct receptors derived from the TrkB gene locus, cDNA cloning and reverse transcription-PCR analysis were used to further analyze receptor isoform expression in the retina and tectum. Receptor isoforms were sequenced that contained a deletion of the N terminus, a deletion in the putative ligand-binding domain, or a deletion in the cytoplasmic juxtamembrane (JM) domain. Two novel JM insertion sequences also were identified, one of which exhibits weak homology to beta-actin and was found in both kinase-containing (TK+) and kinase deletion (KD) receptor isoforms. In the developing retina, TK+ receptor mRNA is upregulated during the period of retinal ganglion cell (RGC) death, consistent with the proposed role of BDNF as a tectal-derived survival factor for RGCs. However, the expression of TK+ transcripts in the tectum indicates that this structure also contains cells responsive to BDNF throughout development. Because BDNF is expressed in both the retina and tectum, it is conceivable that TrkB also mediates autocrine/paracrine signaling within these structures or anterograde retinotectal trophic support.

Aging↗