Structures of homologous composite transposons carrying cbaABC genes from Europe and North America

IS1071 is a class II transposable element carrying a tnpA gene related to the transposase genes of the Tn3 family. Copies of IS1071 that are conserved with more than 99% nucleotide sequence identity have been found as direct repeats flanking a remarkable variety of catabolic gene sequences worldwide. The sequences of chlorobenzoate catabolic transposons found on pBRC60 (Tn5271) in Niagara Falls, N.Y., and on pCPE3 in Bologna, Italy, show that these transposons were formed from highly homologous IS1071 and cbaABC components (levels of identity, > 99.5 and > 99.3%, respectively). Nevertheless, the junction sequences between the IS1071L and IS1071R elements and the internal DNA differ by 41 and 927 bp, respectively, suggesting that these transposons were assembled independently on the two plasmids. The formation of the right junction in both transposons truncated an open reading frame for a putative aryl-coenzyme A ligase with sequence similarity to benzoate- and p-hydroxybenzoate-coenzyme A ligases of Rhodopseudomonas palustris.     

Structural organization of virulence-associated plasmids of Yersinia pestis

The complete nucleotide sequence and gene organization of the three virulence plasmids from Yersinia pestis KIM5 were determined. Plasmid pPCP1 (9,610 bp) has a GC content of 45.3% and encodes two previously known virulence factors, an associated protein, and a single copy of IS100. Plasmid pCD1 (70,504 bp) has a GC content of 44.8%. It is known to encode a number of essential virulence determinants, regulatory functions, and a multiprotein secretory system comprising the low-calcium response stimulation that is shared with the other two Yersinia species pathogenic for humans (Y. pseudotuberculosis and Y. enterocolitica). A new pseudogene, which occurs as an intact gene in the Y. enterocolitica and Y. pseudotuberculosis-derived analogues, was found in pCD1. It corresponds to that encoding the lipoprotein YlpA. Several intact and partial insertion sequences and/or transposons were also found in pCD1, as well as six putative structural genes with high homology to proteins of unknown function in other yersiniae. The sequences of the genes involved in the replication of pCD1 are highly homologous to those of the cognate plasmids in Y. pseudotuberculosis and Y. enterocolitica, but their localization within the plasmid differs markedly from those of the latter. Plasmid pMT1 (100,984 bp) has a GC content of 50.2%. It possesses two copies of IS100, which are located 25 kb apart and in opposite orientations. Adjacent to one of these IS100 inserts is a partial copy of IS285. A single copy of an IS200-like element (recently named IS1541) was also located in pMT1. In addition to 5 previously described genes, such as murine toxin, capsule antigen, capsule anchoring protein, etc., 30 homologues to genes of several bacterial species were found in this plasmid, and another 44 open reading frames without homology to any known or hypothetical protein in the databases were predicted.     

Antigenicity of poly(dA-dT) . poly(dA-dT) photocrosslinked with 8-methoxypsoralen

Hepatitis C virus (HCV) shows substantial nucleotide sequence diversity distributed throughout the viral genome, with many variants showing only 68-79% overall sequence homology. This has led to problems in diagnosis of HCV using commercial immunoassays. Based on clustering of homologous sequences, various genotypes and subtypes of HCV have been described from different geographical regions. In the present study, 11 isolates from India were genotyped using sequence comparison for part of the non-structural (NS5) and structural (core) regions. Parts of the genome covering 451 bp (nt 9-459) of the core gene and a 249 bp fragment (nt 7959-8207) of the NS5 gene were reverse transcribed and amplified using nested polymerase chain reaction (RT-PCR). The amplified fragments were cloned and sequenced. The classification into genotypes was done on the basis of phylogenetic analysis. Four isolates showed sequence homology to type 1b. Two of the isolates were classified as type 3a. One isolate was classified as type 3b and the remaining four isolates were found to be variants of type 3 but did not belong to any designated subtype. On the basis of phylogenetic analysis two of the unclassified isolates were put into a new subtype of 3 named as 3g. In one of these variants, parts of a 5'-noncoding (5' NCR; 204 bp), envelope-E1 (435 bp), and NS3 (502 bp) regions were also amplified, cloned, and sequenced. This study demonstrates the type 3 variants including a new subtype (3g) to be the major cause of HCV infection in India.     

Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria

A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.     

IS1634, a novel insertion element creating long, variable-length direct repeats which is specific for Mycoplasma mycoides subsp. mycoides small-colony type

A new insertion sequence, IS1634, has been identified in Mycoplasma mycoides subsp. mycoides small-colony type (SC). IS1634 shows structural and functional similarities to IS1549 of Mycobacterium smegmatis and with it seems to form a new class or family of insertion sequences. IS1634 has a size of 1,872 bp, including two 13-bp terminal inverted repeats. It contains an open reading frame (ORF) encoding a product of 533 amino acids which shows similarity to the transposase of IS1549 and to a lesser extent to the transposases of IS elements of the IS4 family. IS1634 is present at about 30 copies in the genome of all 22 different field strains of M. mycoides subsp. mycoides SC tested. Characteristic of IS1634 are the long and variable-length direct repeats at the sites of insertion which were found to reach up to about 500 bp. IS1634 is specific to M. mycoides subsp. mycoides SC and is not present in any of the other members of the M. mycoides cluster. Neither was it found in other closely related Mycoplasma species of ruminants.     

Isolation and characterization of cytosolic copper/zinc superoxide dismutase from Capsicum annuum L

A cDNA clone for a cytosolic Cu/Zn superoxide dismutase (Cu/ZnSOD) was isolated and characterized from red pepper (Capsicum annuum L.). The clone consisted of 735 bp containing one open reading frame (ORF) of 459 bp, 46 bp of 5'- and 230 bp of 3'-untranslated region. The nucleotide sequence of the ORF showed 93% homology with that of Nicotiana plumbaginifolia and tomato. It encodes a polypeptide of 154 amino acids with a molecular weight of 15,300. Genomic Southern hybridization suggested that only one copy is present. During cold treatment at 4 degrees C, its expression was maintained at a similar level regardless of illumination.     

Sequence analysis of a 37.6 kbp cosmid clone from the right arm of Saccharomyces cerevisiae chromosome XII, carrying YAP3, HOG1, SNR6, tRNA-Arg3 and 23 new open reading frames, among which several homologies to proteins involved in cell division control and to mammalian growth factors and other animal proteins are found

The nucleotide sequence of 37,639 bp of the right arm of chromosome XII has been determined. Twenty-five open reading frames (ORFs) longer than 300 bp were detected, two of which extend into the flanking cosmids. Only two (L2931 and L2961) of the 25 ORFs correspond to previously sequenced genes (HOG1 and YAP3, respectively). Another ORF is distinct from YAP3 but shows pronounced similarity to it. About half of the remaining ORFs show similarity to other genes or display characteristic protein signatures. In particular, ORF L2952 has striking homology with the probable cell cycle control protein crn of Drosophila melanogaster. L2949 has significant similarity to the human ZFM1 (related to a potential suppressor oncogene) and mouse CW17R genes, though it lacks the carboxy-terminal oligoproline and oligoglutamine stretches encoded by these mammalian genes. The small ORF L2922 is similar to part of the much larger yeast flocculation gene FLO1. Other sequences found in the 37639 bp fragment are one delta and one solo-sigma element, the tRNA-Arg3 gene, the small nuclear RNA gene SNR6 and three ARS consensus sequences.     

Cutaneous suspension: immediate breast reconstruction with abdominal cutaneous advancement using a non-resorptive mesh. Preliminary results and report of 28 cases]

The nucleotide sequence of 22,803 bp on the left arm of chromosome VII was determined by polymerase chain reaction-based approaches to compensate for the unstable character of cosmid clones from this region of the chromosome. The coding density of the sequence is particularly high (more than 83%). Twelve open reading frames (ORFs) longer than 300 bp were found, two of which (at the left side) have been described previously (James et al., 1995) after sequencing of an overlapping cosmid. Four other ORFs correspond to published sequences of the known genes ARO2, RPL9A, TIP1 and MRF1. ARO2 codes for chorismate synthetase. RPL9A for protein L9 of the large ribosomal subunit and MRF1 for a mitochondrial translation release factor. The TIP1 product interacts with Sec20p and is thus involved in transport from endoplasmic reticulum to Golgi. Five of the remaining ORFs have not been identified previously, while the sixth (YGL142c) has been partially sequenced as it lies 5' upstream of MRF1. These six ORFs are relatively large (between 933 and 3657 nucleotides). YGL146c, YGL142c, YGL140c and YGL139w have no significant homology to any protein sequence presently available in the public databases, but show two, nine, nine and eight putative transmembrane spans, respectively. YGL144c has a serine active site signature of lipases. YGL141w has limited homology to several human proteins, one of which mediates complex formation between papillomavirus E6 oncoprotein and tumor suppressor protein p53.     

An analysis of a class of DNA sequence reading molecules

The bacterial insertion sequence IS21 contains two genes, istA and istB, which are organized as an operon. IS21 spontaneously forms tandem repeats designated (IS21)2. Plasmids carrying (IS21)2 react efficiently with other replicons, producing cointegrates via a cut-and-paste mechanism. Here we show that transposition of a single IS21 element (simple insertion) and cointegrate formation involving (IS21)2 result from two distinct non-replicative pathways, which are essentially due to two differentiated IstA proteins, transposase and cointegrase. In Escherichia coli, transposase was characterized as the full-length, 46 kDa product of the istA gene, whereas the 45 kDa cointegrase was expressed, in-frame, from a natural internal translation start of istA. The istB gene, which could be experimentally disconnected from istA, provided a helper protein that strongly stimulated the transposase and cointegrase-driven reactions. Site-directed mutagenesis was used to express either cointegrase or transposase from the istA gene. Cointegrase promoted replicon fusion at high frequencies by acting on IS21 ends which were linked by 2, 3, or 4 bp junction sequences in (IS21)2. By contrast, cointegrase poorly catalyzed simple insertion of IS21 elements. Transposase had intermediate, uniform activity in both pathways. The ability of transposase to synapse two widely spaced IS21 ends may reside in the eight N-terminal amino acid residues which are absent from cointegrase. Given the 2 or 3 bp spacing in naturally occurring IS21 tandems and the specialization of cointegrase, the fulminant spread of IS21 via cointegration can now be understood.     

Cloning and characterization of the lactococcal plasmid-encoded type II restriction/modification system, LlaDII

The LlaDII restriction/modification (R/M) system was found on the naturally occurring 8.9-kb plasmid pHW393 in Lactococcus lactis subsp. cremoris W39. A 2.4-kb PstI-EcoRI fragment inserted into the Escherichia coli-L. lactis shuttle vector pCI3340 conferred to L. lactis LM2301 and L. lactis SMQ86 resistance against representatives of the three most common lactococcal phage species: 936, P335, and c2. The LlaDII endonuclease was partially purified and found to recognize and cleave the sequence 5'-GC decreases NGC-3', where the arrow indicates the cleavage site. It is thus an isoschizomer of the commercially available restriction endonuclease Fnu4HI. Sequencing of the 2.4-kb PstI-EcoRI fragment revealed two open reading frames arranged tandemly and separated by a 105-bp intergenic region. The endonuclease gene of 543 bp preceded the methylase gene of 954 bp. The deduced amino acid sequence of the LlaDII R/M system showed high homology to that of its only sequenced isoschizomer, Bsp6I from Bacillus sp. strain RFL6, with 41% identity between the endonucleases and 60% identity between the methylases. The genetic organizations of the LlaDII and Bsp6I R/M systems are identical. Both methylases have two recognition sites (5'-GCGGC-3' and 5'-GCCGC-3') forming a putative stemloop structure spanning part of the presumed -35 sequence and part of the intervening region between the -35 and -10 sequences. Alignment of the LlaDII and Bsp6I methylases with other m5C methylases showed that the protein primary structures possessed the same organization.     

Expression and regulation of constitutive and acute phase serum amyloid A mRNAs in hepatic and non-hepatic cell lines

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.     

Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2

The genome of the transposable element IS2 contains five open reading frames that are capable of encoding proteins greater than 50 amino acids; however, only one IS2 protein of 14 kDa had been detected. By replacing the major IS2 promoter located in the right terminal repeat of IS2 with the T7 promoter to express IS2 genes, we have detected another IS2 protein of 46 kDa. This 46-kDa protein was designated InsAB'. Analyses of the InsAB' sequence revealed motifs that are characteristic of transposases of other transposable elements. InsAB' has the ability to bind both terminal repeat sequences of IS2. It was shown to bind a 27-bp sequence (5'-GTTAAGTGATAACAGATGTCTGGAAAT-3', positions 1316 to 1290 by our numbering system [16 to 42 by the previous numbering system]) located at the inner end of the right terminal repeat and a 31-bp sequence (5'-TTATTTAAGTGATATTGGTTGTCTGGAGATT-3', positions 46 to 16 [1286 to 1316]), including the last 27 bp of the inner end and the adjacent 4 bp of the left terminal repeat of IS2. This result suggests that InsAB' is a transposase of IS2. Since there is no open reading frame capable of encoding a 46-kDa protein in the entire IS2 genome, this 46-kDa protein is probably produced by a translational frameshifting mechanism.     

ART-CH: a VL30 in chickens?

The complete sequence of ART-CH, a recently found chicken retrotransposon (A. V. Gudkov, E. A. Komarova, M. A. Nikiforov, and T. E. Zaitsevskaya, J. Virol. 66:1726-1736, 1992), was characterized. ART-CH has the structure of a 3,300-bp-long provirus, including two 388-bp long terminal repeats (LTRs) (U3, 245 bp; R region, 17 bp; and U5, 126 bp), a tRNA(Trp)-binding site, and a polypurine tract, similar to avian leukosis viruses. At least some of the approximately 50 genomic copies of ART-CH are transcribed into polyadenylated RNA, which is initiated and terminated at the expected sites within the LTRs. In contrast to the regulatory sequences involved in proviral expression and replication, the internal regions of ART-CH seem to be completely defective. Several short regions of homology with avian leukosis virus genes, most of which encode gag-related sequences, were found among different reading frames of ART-CH, which are not organized like regular retroviral genes. Both sequence analysis and restriction fragment length polymorphism analysis revealed a high degree of sequence (97% homology) and structural similarity among members of the ART-CH family, indicating their common origin and recent penetration into chicken DNA. ART-CH sequences were detected in mouse cells infected with Rous sarcoma virus produced by an ART-CH-expressing Rous sarcoma. These data are consistent with the hypothesis that ART-CH belongs to a class of defective retrotransposons whose replication strategy requires the use of helper viruses. They might originate from an avian leukosis virus-related retrovirus which completely lost its coding capacities as a result of multiple mutations and deletions. These features apparently group ART-CH with the VL30 retrotransposons of rodents.     

Polymerase chain reaction in the diagnosis of tuberculosis. Comparison of two target sequences for amplification

Amplification of a 340 bp sequence of the 38 kDa protein gene of Mycobacterium tuberculosis by the polymerase chain reaction has been developed. The sensitivity of this PCR was shown to be 10 fg both by agarose gel electrophoresis and Southern blot hybridisation being equivalent to 2-3 organisms and highly specific to M. tuberculosis and excluding even M. tuberculosis H37Ra and Mycobacterium bovis BCG. Sputum samples from patients with pulmonary tuberculosis gave a positivity rate of 45%. PCR was also performed using pt8 and pt9 primers which amplified a 541 bp sequence of IS6110. 41% of the above samples gave positive amplification. Three samples that were positive for 38 kDa sequence were negative for IS6110.