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PubMed2002

Novel splice variants of amphiphysin I are expressed in retina.

Using RT-PCR-based cDNA cloning, we identified novel splice variants of amphiphysin I, termed amph Ir, that are expressed specifically in retina. In comparison with the prototype amphiphysin I, amph Ir contained two novel insertions (inserts A and B) and one deletion. Insert A is only 9 bp in length but appears to be a determinant for the retina-specific expression. In contrast, insert B is a large domain of 1740 bp and two shorter transcripts with 3'-truncated insert B were also expressed. All the insert sequences were present as unidentified exons in the amphiphysin I gene on human chromosome 7. Western blot analysis of various rat tissues with anti-insert B antibody confirmed the presence and tissue specificity of the variant proteins.

Alternative Splicing↗

PubMed2003

cDNA cloning of thyroid hormone receptor betas from the conger eel, Conger myriaster.

Two distinct TRbetas cDNA clones were generated by RACE from a conger eel (Conger myriaster). The deduced amino acid sequences of the conger eel TRbetas (cTRbetas) showed higher homologies to the known TRbetas of other vertebrate animals than to TRalphas. Two cTRbetas possessed an insertion sequence in the hinge region similar to other teleost fish TRbetas. Variation in cTRbeta due to differential splicing in the hinge region of the cTRbetas genes was found using PCR analysis. The determination of TRbeta mRNA levels in eel tissue was performed using competitive RT-PCR. The cTRbeta1 mRNA was widely expressed, whereas cTRbeta 2 mRNA was most highly expressed in the brain and pituitary. The expression pattern of cTRbeta1 and cTRbeta2 in tissues were similar to that of TRbeta1 and TRbeta2 in mammals.

Amino Acid Sequence↗

PubMed2003

DNA binding and telomere length regulation of yeast RAP1 homologues.

The repressor activator protein 1 (RAP1) has many important functions in Saccharomyces cerevisiae. At the chromosome ends, it is a negative regulator of telomere length. Here, we show that Saccharomyces castellii/Saccharomyces dairensis telomeric sequences inserted into a S.cerevisiae telomere are counted as part of the telomere, consistent with the presence of high-affinity Rap1p binding sites within these sequences. We show that S.castellii Rap1p (scasRap1p) can regulate telomere length in a S.cerevisiae strain, albeit less stringently. Cloning of the S.dairensis RAP1 homologue (sdaiRAP1) revealed that it encodes the largest RAP1 protein identified to date. Despite its large size, binding analyses of the recombinant sdaiRap1p revealed that the protein binds with the same spacing and with similar affinity to yeast telomeric sequences, as the scer- and scasRAP1 proteins. According to the Rap1p counting model for telomere length regulation, a low density of Rap1p binding sites in a telomere would result in a longer telomere in S.cerevisiae. We have compared the lengths of two individual S.dairensis telomeres and find that their lengths are not regulated to give the same number of high-affinity binding sites. This may be due to other factors than Rap1p having influence on the telomere length regulation.

Animals↗

PubMed1985

Sequence analysis of the glyW region in Escherichia coli.

We have determined the DNA sequence of a 1-kilobase segment of the Escherichia coli chromosome. The segment contains glyW, a duplicate gene for tRNA3Gly, and its flanking regions. An insertion sequence, previously known to have occurred spontaneously within the sequenced fragment, was identified as IS1. Possible sites for initiation and termination of transcription were identified by comparing them with the sequences of model promoter regions and termination structures. The results suggest that the expression of glyW may depend upon the expression of the preceding gene, pgsA, by transcriptional or translational overlap, by cotranscription of these two genes, or both.

Amino Acid Sequence↗

PubMed1999

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

Cell-Free System↗

PubMed2001

Epidemiology of the genetic elements responsible for acquired glycopeptide resistance in enterococci.

Five genotypes of acquired glycopeptide resistance have been documented in enterococci, with vanA and vanB being the most globally widespread and prevalent. Resistance results from the production of peptidoglycan precursors with reduced binding affinity for glycopeptides and is encoded by complex clusters of van genes. The prototype VanA element is Tn1546, a 10.8-kb transposon that carries the vanRSHAXYZ genes. Diverse VanA elements exist, but all share the vanRSHAX cluster and are believed to be derived from a Tn1546 progenitor. The sequences of these genes are remarkably conserved, with only a few point mutations identified. VanA elements do however vary by the presence of deletions and insertion sequences (IS) in nonessential genes (orf1, orf2, vanY, and vanZ) and intergenic regions. IS transposition probably plays a key role in VanA element evolution. By contrast, vanB gene clusters show greater sequence divergence. Three vanB alleles have been reported, of which vanB2 appears to be the most widespread, generally as part of Tn5382 and related elements. To date, only four Enterococcus faecium strains with VanD resistance have been reported, and each contained a distinct vanD allele. The VanE and VanG types have each been identified in single strains of Enterococcus faecalis. The existence of distinct genotypes, together with the allelic nature of vanB and vanD, suggests that van clusters have transferred to enterococci on multiple occasions from undefined donor species, with subsequent horizontal dissemination, particularly of VanA and VanB elements, among enterococci. Characterization of glycopeptide resistance elements yields information on their evolution and broadens our insights into the epidemiology of resistant enterococci.

Anti-Bacterial Agents↗

PubMed1997

DNase I primary transcript is alternatively spliced in both normal and apoptotic cells: no evidence of up-regulation in apoptosis.

The reverse transcriptase polymerase chain reaction (RT-PCR) was used to elucidate the expression of DNase I and its possible involvement in apoptotic genome degradation. Multiple PCR products were obtained from cDNAs of different rat and human cells. The subsequent cloning and sequence analysis of seven PCR products revealed that only one of them had the expected size (639 bp) and sequence identity to that of rat DNase I cDNA. The other six PCR products were characterized by either sequence insertions or deletions. To establish the origin of this molecular diversity, a genomic fragment of the rat DNase I gene was also isolated, subcloned, and sequenced. Sequence comparison of six cDNAs with the rat genomic DNA revealed that they, indeed, resulted from inclusion of introns or deletion of exons. Southern hybridizations of the RT-PCR products from a variety of mammalian cell lines, using the major DNase I cDNA fragment as a probe, showed that in some cells as many as 20 alternatively spliced transcripts could be detected. This complexity of splice variants was widespread, and cell-specific profiles differed by the relative concentration of each transcript. None of these spliced transcripts maintained an open reading frame containing an intact catalytic site, suggesting that they do not encode any functional proteins. These complicated alternative splicing events might, however, significantly contribute to the regulation of DNase I expression. There was no apparent increase of the major DNase I transcript after induction of apoptosis in the cell lines studied. Apoptotic cells appeared to have similar normal/alternative transcript ratios as the control cells, suggesting that DNase I may not be the endonuclease involved in DNA degradation during apoptosis.

3T3 Cells↗

PubMed2004

Functional identification of rice syn-copalyl diphosphate synthase and its role in initiating biosynthesis of diterpenoid phytoalexin/allelopathic natural products.

Rice produces a number of phytoalexins, and at least one allelopathic agent, from syn-copalyl diphosphate (CPP), representing the only known metabolic fate for this compound. Thus, the class II terpene synthase that converts the universal diterpenoid precursor geranylgeranyl diphosphate to syn-CPP catalyzes the committed step in biosynthesis of these natural products. Here the extensive sequence information available for rice was coupled to recombinant expression and functional analysis to identify syn-copalyl diphosphate synthase (OsCPSsyn). In addition, OsCPSsyn mRNA was found to be specifically induced in leaves by conditions that stimulate phytoalexin biosynthesis. Therefore, transcription of OsCPSsyn seems to be an important regulatory point for controlling the production of these defensive compounds. Finally, alignments carried out with OsCPSsyn revealed that class II terpene synthases exhibit a sequence conservation pattern substantially different from that of the prototypical class I enzymes. One particularly notable feature is the specific conservation of the functionally cryptic 'insertional' sequence element in class II terpene synthases, indicating that this region is important for the corresponding cyclization reaction.

Alkyl and Aryl Transferases↗

PubMedDate not supplied

A short internal eliminated sequence with central conserved sequences interrupting the LA-MSC gene of the 81 locus in the hypotrichous ciliates Oxytricha fallax and O. trifallax.

IES-LA is a short Internal Eliminated Sequence interrupting LA-MSC, a protein-coding gene of the 81 locus of Oxytricha fallax and O. trifallax. IES-LA is precisely excised from the gene during development of the macronucleus. The internal eliminated sequence is bounded by CAAT ... AATG, and thereby resembles a TBE1 transposon internal eliminated sequence insertion that is grossly shortened (4.1 kbp to 52-64 bp), consistent with the hypothesis that short IESs are degenerated ancient transposons. The pattern of sequence conservation between five alleles of IES-LA shows that it differs from previously characterized classes of ciliate short IESs: while many short IESs have conserved ends and diverged centers, IES-LA is more conserved in its center and its ends are diverged. This implies a excision mechanism for IES-LA that is distinct from those for other known Oxytricha IESs.

Animals↗

PubMed1997

Enterococcal transposon Tn5384: evolution of a composite transposon through cointegration of enterococcal and staphylococcal plasmids.

Mechanisms for the possible transfer of antimicrobial resistance genes between staphylococci and enterococci remain poorly defined. We have previously reported the transfer between Enterococcus faecalis strains of a multiresistance chromosomal element (beta-lactamase positive and resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, and tetracycline) which we have tentatively designated Tn5385. Tn5385 is a composite of several smaller transposable elements, including Tn5384, a 26-kb composite transposon conferring resistance to erythromycin, gentamicin, and mercuric chloride. Analyses of 7 kb within Tn5384 and flanking sequences within the larger element revealed sequences characteristic of staphylococcal beta-lactamase and small, mobilizable plasmids flanking a region with a sequence identical to those of the replication genes previously described for enterococcal and streptococcal broad-host-range plasmids. These diverse regions are linked by insertion sequences IS256 and IS257 in a manner which suggests a series of cointegration events as the genesis of the current relationship. Taken together, these data suggest that Tn5384 and the larger element within which it is incorporated (Tn5385) evolved at least in part as a result of cointegration between an enterococcal broad-host-range plasmid and staphylococcal beta-lactamase and small mobilizable plasmids. These results implicate broad-host-range plasmids in the transfer of resistance determinants from staphylococci to enterococci.

Base Sequence↗

PubMed2002

Novel complex sul1-type integron in Escherichia coli carrying bla(CTX-M-9).

For the present report, a novel complex class 1 integron, In60, was characterized. Part of this integron includes the bla(CTX-M-9) gene and its downstream nucleotide sequence, which shares 81% and 78% nucleotide identity with those of kluA-1 beta-lactamase and orf3 of K. ascorbata, respectively. Furthermore, a new insertion sequence, IS3000, has been found in In60. PCR analysis indicates that integron In60 is present in 33 of 34 nonclonal enterobacterial isolates carrying the putative beta-lactamase CTX-M-9.

Base Sequence↗

PubMed2002

Genome sequence of Yersinia pestis KIM.

We present the complete genome sequence of Yersinia pestis KIM, the etiologic agent of bubonic and pneumonic plague. The strain KIM, biovar Mediaevalis, is associated with the second pandemic, including the Black Death. The 4.6-Mb genome encodes 4,198 open reading frames (ORFs). The origin, terminus, and most genes encoding DNA replication proteins are similar to those of Escherichia coli K-12. The KIM genome sequence was compared with that of Y. pestis CO92, biovar Orientalis, revealing homologous sequences but a remarkable amount of genome rearrangement for strains so closely related. The differences appear to result from multiple inversions of genome segments at insertion sequences, in a manner consistent with present knowledge of replication and recombination. There are few differences attributable to horizontal transfer. The KIM and E. coli K-12 genome proteins were also compared, exposing surprising amounts of locally colinear "backbone," or synteny, that is not discernible at the nucleotide level. Nearly 54% of KIM ORFs are significantly similar to K-12 proteins, with conserved housekeeping functions. However, a number of E. coli pathways and transport systems and at least one global regulator were not found, reflecting differences in lifestyle between them. In KIM-specific islands, new genes encode candidate pathogenicity proteins, including iron transport systems, putative adhesins, toxins, and fimbriae.

Bacteriophages↗

PubMed1991

Cloning, sequencing and expression of the sialidase gene from Actinomyces viscosus DSM 43798.

Chromosomal DNA from Actinomyces viscosus was digested with restriction endonucleases and the fragments ligated with pUC-vectors were used to transform Escherichia coli cells. Clones bearing the required sialidase gene were detected by spraying the colonies with the fluorogenic sialidase substrate MU-Neu5Ac. The identity of the cloned sialidase was confirmed after the 5700-fold enrichment and comparison with the purified enzyme of A. viscosus. Both sialidases were identical with regard to molecular mass, substrate specificity tested with sialyllactoses, and the inhibition of their activity by heterologous antisialidase antibodies. The sequenced insert (EMBL accession number X62276) revealed a mol% G + C of 68.2, typical for A. viscosus. An open reading frame of 2739 bp follows a sequence with dyad symmetry and an AG-rich region, and codes for 913 amino acids representing a molecular mass of 113 kDa. The conserved amino acid sequence [Ser-X-Asp-X-Gly-X-Thr-Trp] typical for bacterial sialidases was found at five positions in the predicted amino acid sequence. The gene of this enzyme is expressed by E. coli, despite the low relatedness of both species.

Actinomyces viscosus↗

PubMed1995

A novel insertion element from Mycobacterium avium, IS1245, is a specific target for analysis of strain relatedness.

The insertion sequence IS1245 is a novel mycobacterial repetitive element identified in Mycobacterium avium. It encodes a transposase which exhibits a 64% amino acid similarity with IS1081, an insertion element present in the M. tuberculosis complex. The host range of IS1245 appears limited to M. avium as this element was not identified in M. intracellulare or in any other of 18 mycobacteria species tested. When IS1245 was used for restriction fragment length polymorphism (RFLP) analysis, human isolates characteristically presented a high number of copies (median, 16; range, 3 to 27) and a diversity of RFLP patterns comparable to that found by pulsed-field gel electrophoresis. Isolates from nonhuman sources differed both in number of copies and in RFLP pattern diversity: while swine isolates shared the characteristics of human strains, those from several avian sources exhibited a very low copy number of IS1245 and appeared clonal on the basis of RFLP.

Animals↗

PubMed1997

Isolation and characterization of IS1 circles.

Transposase encoded by insertion sequence IS1 is produced from two out-of-phase reading frames by translational frameshifting that occurs in a run of adenines. An IS1 mutant with a single adenine insertion in the run of adenines efficiently produces transposase, resulting in generation of miniplasmids by deletion for a region adjacent to IS1 from a plasmid carrying the IS1 mutant. Here, we found that besides miniplasmids, cells harboring the plasmid contained minicircles without the region required for replication. Cloning and DNA sequencing of the minicircles revealed that most of them were IS1 circles consisting of the entire IS1 sequence and a sequence, 5-9 bp in length, which intervenes between terminal inverted repeats, IRL and IRR, of IS1. Analysis of more IS1 circles isolated by polymerase chain reaction revealed that the intervening sequence was derived from the region flanking either IRL or IRR in the parental plasmid, suggesting that IS1 circles are generated by an excision event from the parental plasmid. The IS1 circles may be formed due to the cointegration reaction occurring within the parental plasmid carrying IS1.

Cloning, Molecular↗

PubMed1995

IS1-encoded proteins, InsA and the InsA-B'-InsB transframe protein (transposase): functions deduced from their DNA-binding ability.

Insertion sequence IS1 encodes a transframe protein, InsA-B'-InsB, which is produced from two out-of-phase reading frames, insA and B'-insB, by translational frameshifting at a run of adenines. Unless the frameshifting event occurs, the InsA protein is produced from IS1. We found that cells harboring a plasmid carrying an IS1 mutant with a single adenine insertion in the run of adenines contained miniplasmids. Cloning and DNA sequencing analyses of the miniplasmids revealed that they had a deletion extending from an inverted repeat (IR) at the left end of IS1. This indicates that they were generated by IS1-mediated deletion due to efficient production of the InsA-B'-InsB transframe protein that is IS1 transposase. Both the InsA protein and transposase were partially purified as a fusion protein with collagen-LacZ by LacZ-specific affinity column chromatography. The InsA* and the collagenolyzed InsA* were found to bind specifically to a 24-bp region within each of the IRs at the ends of IS1. The transposase Tnp* and the collagenolyzed Tnp* were found to bind to the sequence with or without IR, but preferentially to that with IR. The nonspecific DNA-binding ability of transposase may be involved in recognition of the target DNA, an important process of transposition of IS1. Both InsA and transposase have the IR-specific DNA binding ability and a common polypeptide segment containing the alpha-helix-turn-alpha-helix motif, supporting the previous indication that InsA competes with transposase to bind to IRs and thus becomes a transposition inhibitor. Based on the observations described in this article, we speculate that transposase of IS1 consists of at least two domains, the N-terminal half, which almost entirely overlaps InsA, and the C-terminal half, which almost entirely overlaps B'-InsB. The frameshifting event adds the latter domain to the former to give the transposase activity recognizing IRs and the target sequence to initiate the transposition reaction.

Amino Acid Sequence↗

PubMed1992

Gene disruption identifies a 290 kDa cell-surface polypeptide conferring hydrophobicity and coaggregation properties in Streptococcus gordonii.

The C-terminal coding region of the gene (denoted cshA) encoding a high-molecular-mass (290 kDa) cell-surface polypeptide in the oral bacterium Streptococcus gordonii was cloned and sequenced. Insertion of ermAM into the S. gordonii chromosome at the 3' end of the coding region of cshA led to the production of isogenic mutants that secreted a truncated form (260 kDa) of the CshA polypeptide into the growth medium. Mutants had reduced cell-surface hydrophobicity and were impaired in their ability to coaggregate with oral actinomyces. The results identify a carboxyl terminus-anchored cell-surface protein determinant of hydrophobicity and coaggregation in S. gordonii.

Actinomyces↗

PubMed1990

Stable RNA secondary structure in a retroviral vector insert terminates reverse transcriptase elongation in vitro but not in cultured cells.

We wished to test whether an RNA signal that causes termination of elongation by reverse transcriptase in vitro would affect retroviral vector function. A synthetic oligonucleotide containing a sequence capable of forming a very stable RNA secondary structure was subcloned into the retrovirus vector N2. The integration of this sequence into N2 causes termination of elongation by reverse transcriptase in vitro at the precise positions previously reported in a different sequence context. However, no premature termination of DNA synthesis was observed in the unintegrated DNA of vector transduced cells. Likewise, there was no deleterious effect of the sequence insert on vector titer. These results indicate that termination signals defined in in vitro systems cannot be used as predictors of in vivo function and suggest that viral proteins in addition to reverse transcriptase play an important role in transcript initiation and elongation.

Animals↗