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PubMed1998

A software system for data analysis in automated DNA sequencing.

Software for gel image analysis and base-calling in fluorescence-based sequencing consisting of two primary programs, BaseFinder and GelImager, is described. BaseFinder is a framework for trace processing, analysis, and base-calling. BaseFinder is highly extensible, allowing the addition of trace analysis and processing modules without recompilation. Powerful scripting capabilities combined with modularity and multilane handling allow the user to customize BaseFinder to virtually any type of trace processing. We have developed an extensive set of data processing and analysis modules for use with the program in fluorescence-based sequencing. GelImager is a framework for gel image manipulation. It can be used for gel visualization, lane retracking, and as a front end to the Washington University Getlanes program. The programs were designed using a cross-platform development environment, currently allowing them to run in Windows NT, Windows 95, Openstep/Mach, and Rhapsody. Work is ongoing to deploy the software on additional platforms, including Solaris, Linux, and MacOS. This software has been thoroughly tested and debugged in the analysis of >2 million bp of raw sequence data from human chromosome 19 region q13. Overall sequencing accuracy was measured using a significant subset of these data, consisting of approximately 600 sequences, by comparing the individual shotgun sequences against the final assembled contigs. Also, results are reported from experiments that analyzed the accuracy of the software and two other well-known base-calling programs for sequencing the M13mp18 vector sequence. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF025422]

Algorithms↗
PubMed2002

Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species.

A range of gram-negative bacterial species use N-acyl homoserine lactone (AHL) molecules as quorum-sensing signals to regulate different biological functions, including production of virulence factors. AHL is also known as an autoinducer. An autoinducer inactivation gene, aiiA, coding for an AHL lactonase, was cloned from a bacterial isolate, Bacillus sp. strain 240B1. Here we report identification of more than 20 bacterial isolates capable of enzymatic inactivation of AHLs from different sources. Eight isolates showing strong AHL-inactivating enzyme activity were selected for a preliminary taxonomic analysis. Morphological phenotypes and 16S ribosomal DNA sequence analysis indicated that these isolates probably belong to the species Bacillus thuringiensis. Enzymatic analysis with known Bacillus strains confirmed that all of the strains of B. thuringiensis and the closely related species B. cereus and B. mycoides tested produced AHL-inactivating enzymes but B. fusiformis and B. sphaericus strains did not. Nine genes coding for AHL inactivation were cloned either by functional cloning or by a PCR procedure from selected bacterial isolates and strains. Sequence comparison of the gene products and motif analysis showed that the gene products belong to the same family of AHL lactonases.

4-Butyrolactone↗
PubMed2002

[Study on the cloning, expression and the immunogenicity of helicobacter pylori heat shock protein 60 gene].

OBJECTIVE: To construct a recombinant strain of bacteria expressing heat shock protein of (Hsp) Helicobacter pylori (Hp) and study the immunogenicity of Hsp60. METHODS: PCR amplification of Hsp60 DNA was performed before it was inserted into the prokaryotic expression vector pET-22b(+) to transform BL21(DE3) E.coli strain. Hsp60 expressed by the recombinant E.coli was collected and purified for immunogenicity assessment in mice. RESULTS: DNA sequence analysis showed identical DNA sequence of Hsp60 thus produced to that published in Genbank. Accounting for a ratio of 27.2% among the total protein production in the bacterium, recombinant Hsp60 protein was recognized by the serum from Hp-infected patients and produced corresponding antibody in Balb/c mice in response to immunization. CONCLUSION: Recombinant Hsp60 protein can be used potentially as a vaccine for controlling and treating Hp infection.

Bacterial Proteins↗
PubMed1996

Molecular analysis of lacI mutations in Rat2 cells exposed to 7,12-dimethylbenz[a]anthracene: evidence for DNA sequence and DNA strand biases for mutation.

The Rat2 cell line carries 50-70 stably integrated copies per cell of a lambda/lacI shuttle vector as a target for mutagenicity testing. Rat2 cells were exposed to 1 and 10 micrograms/ml of 7,12-dimethylbenz[a]anthracene (DMBA) for 24 h at 37 degrees C in the presence of primary rat hepatocytes, and grown to confluence. The shuttle vector was rescued from untreated and mutagen-treated cells and mutant frequencies were determined. The low and high doses of DMBA induced mutant frequencies that were 7-fold (25 +/- 4.9 x 10(-5)) and 33-fold (127 +/- 19.9 x 10(-5)) higher, respectively, than the spontaneous mutant frequency (3.8 +/- 0.7 x 10(-5)). DNA sequence analysis of the DMBA-induced lacI- mutants indicated that they contained mainly basepair substitution mutations at A:T and G:C, and that A:T-->T:A and G:C-->T:A transversions were the predominant types. In addition, 23 of 28 (82%) A:T basepair substitution mutations occurred with the mutated dA, the putatively adducted base, on the coding strand. Furthermore, 20 of the 28 (71%) A:T mutations had the mutated dA flanked 5' by a dC, and 17 of these were A:T-->T:A transversions, suggesting a sequence preference for this mutation. Except for a higher proportion of G:C-->A:T transitions in the low dose data, the mutational profiles from low and high doses of DMBA were similar. These results indicate that DMBA mutagenesis in the lacI gene of Rat2 cells displays distinct DNA sequence and DNA strand preferences.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Structural analysis of the mouse T-cell receptor Tcra V2 subfamily.

Cosmid clones containing T-cell receptor Tcra V2 subfamily gene segments have been isolated from a BALB/c cosmid library and subjected to DNA sequence analysis. The V gene segments in the Tcra V2 subfamily differ from each other by 3%-7% at the nucleotide level and 5%-16% at the amino acid level. T-cell receptor Tcra V2 gene segment polymorphisms have been identified in the B10.PL and PL/J mouse strains with a Tcra V2 subfamily-specific probe. These V gene segment polymorphisms may cause the differential Tcra V gene usage in induced experimental allergic encephalomyelitis between B10.PL and PL/J mice.

Amino Acid Sequence↗
PubMed2003

Relationship between nosocomial Acinetobacter species occurring in two geographical areas (Greece and the UK).

Fifty-two isolates of Acinetobacter spp. obtained from three Greek and one UK hospital, were studied using partial 16 S ribosomal DNA sequence analysis, repetitive extragenic palindromic sequence-based polymerase chain reaction (REP-PCR) mediated fingerprinting and DNA macro-restriction analysis. The aim was twofold: first, to discern the major differences in the population of Acinetobacter spp. between the two countries. Second, to compare a simple PCR-based typing scheme with pulsed-field gel electrophoresis (PFGE). The multi-resistant Greek isolates were within DNA groups 2 and TU13, and clustered into three types both by REP-PCR and PFGE. By contrast, the more susceptible Oxford isolates were heterogeneous on 16 S RNA sequence analysis and distinguishable on typing. The need for studies that elucidate the phylogeny of Acinetobacter spp. inside and outside hospitals are important, as this will help clarify the relationship between organisms that are increasingly recognized as causes of severe infections.

Acinetobacter Infections↗
PubMed1994

A new subtype of human immunodeficiency virus type 1 (MVP-5180) from Cameroon.

A new subtype (MVP-5180) of human immunodeficiency virus type 1 (HIV-1) was isolated from a Cameroonian AIDS patient. MVP-5180 was grown in several human T-cell lines and the monocytic U937 line. MVP-5180 DNA could not be amplified by nested primer PCR with conventional env primers and could be only very faintly amplified with gag and pol primers. Most German, Ivoirian, and Malawian anti-HIV-1 sera reacted faintly or moderately with Env proteins in an MVP-5180 immunoblot, whereas some Cameroonian sera reacted strongly. Of HIV-1-infected Cameroonians, 8% were identified by serological methods as infected with MVP-5180; 7% were positive when MVP-5180-specific PCR env primers were used. DNA sequence analysis of MVP-5180 showed that its genetic organization was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP-5180 had similarities to HIV-1 and HIV-2 of 53 and of 49%, respectively. V3 loop analysis identified a crown of Gly-Pro-Met-Arg by using cloned DNA and Gly-Pro-Leu-Arg by using PCR-amplified DNA, neither of which configuration has been described for other HIV strains. In an analysis of relationships, MVP-5180 occupied a position distant to all other HIV-1 strains, including the chimpanzee simian immunodeficiency virus type 1 SIVcpz and the Uganda virus U455, and closer to the HIV-1/HIV-2 divergence node. MVP-5180, together with another Cameroonian isolate, ANT-70, constitutes a group subtype O of the most divergent HIV-1 isolates yet identified. Characterization of MVP-5180 is important for understanding the natural history of the primate immunodeficiency viruses and for the development of vaccines and diagnostics.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Polyphasic characterisation of Burkholderia cepacia-like isolates leading to the emended description of Burkholderia pyrrocinia.

Twenty-five Burkholderia cepacia-like isolates of human and environmental origin, comprising five different recA RFLP types, were examined by using a polyphasic taxonomic approach, including recA gene sequence analysis, 16S rRNA gene sequence analysis, DNA:DNA hybridisation studies, tDNA-PCR, fatty acid analysis and biochemical analysis. The results of the present study demonstrated that twenty-three of these strains belong to Burkholderia pyrrocinia, a B. cepacia complex species thus far comprising one single soil isolate only. An emended description of Burkholderia pyrrocinia is proposed. The taxonomic status of the remaining two isolates requires further analysis.

Bacterial Typing Techniques↗
PubMed1994

Genetic analysis of human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) mixed infections in India reveals a recent spread of HIV-1 and HIV-2 from a single ancestor for each of these viruses.

DNA sequences encoding the surface envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) were amplified by PCR from uncultured peripheral blood mononuclear cells obtained from patients with serologically defined HIV-1/HIV-2 mixed infections from Bombay, India. HIV-1-specific PCR products were obtained in seven of seven randomly chosen doubly reactive cases, while HIV-2-specific sequences were detected in five of seven cases (71%). DNA sequence analysis showed that the HIV-1 gp120 coding sequences were closely related to each other (nucleotide sequence divergence of between 3.1 and 6.8%). Phylogenetic tree analysis placed the Indian strains within the C subtype of HIV-1, being most similar to sequences previously found in East and South Africa. The HIV-2 sequences were also closely related to each other, with an overall sequence divergence of between 5.6 and 10.5%. The low level of nucleotide divergence among Indian HIV-1 and HIV-2 sequences suggests a fairly recent introduction of each virus into this population from a single point of entry in each case. The HIV-2 sequences reported here represent the first analysis of Asian HIV-2 strains and confirm the serological pattern previously detected in India. These data show that a substantial spread of HIV-2, together with HIV-1, has appeared outside Africa in a population hitherto unexposed to HIV. These findings imply that further spread of HIV-2 worldwide is to be expected and have important implications for future vaccine and therapy development.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Group II intron from Pseudomonas alcaligenes NCIB 9867 (P25X): entrapment in plasmid RP4 and sequence analysis.

Pseudomonas alcaligenes NCIB 9867 (strain P25X), which grows on 2,5-xylenol and harbours the plasmid RP4, was mated with a plasmid-free derivative of Pseudomonas putida NCIB 9869, strain RA713, which cannot grow on 2,5-xylenol. Some RA713 transconjugants, initially selected on 2,5-xylenol, were found to carry RP4 plasmids that had acquired additional fragments (designated Xin) which ranged in size from 2 kb to approximately 26 kb instability of DNA inserts in RP4::Xin hybrid plasmids was observed. The smallest insert present in a stable RP4::Xin6 hybrid plasmid, termed Xin6, yielded multiple bands when it was used as a probe with digested P25X chromosomal DNA. Sequence analysis of Xin6 led to the discovery of an open reading frame with homology to the maturases of group II introns. The Xin6 insert also exhibited several features characteristic of a group II intron. These included the presence of the consensus sequence GUGYG at the 5' and and RAY at the 3' end of the intron. RNA secondary structure modelling of Xin6 also revealed the presence of perfectly conserved domains V and VI. Differences were detected in the Xin6 hybridization profiles of several P25X catabolic mutants that have lost the ability to grow on 2,5-xylenol. In these mutants the loss of 2,5-xylenol degradative ability could be due to genome rearrangements mediated by sequences related to the Xin6 group II intron. This is the first reported group II intron isolated from Pseudomonas spp. and the first time that the mobility of a bacterial group II intron has been demonstrated.

Amino Acid Sequence↗
PubMed1994

Structure and function of a mating-type gene from the homothallic species Neurospora africana.

The homothallic Neurospora species, N. africana, contains sequences that hybridize to the A but not to a mating-type sequences of the heterothallic species N. crassa. In this study, the N. africana mating-type gene, mt A-1, was cloned, sequenced and its function analyzed in N. crassa. Although N. africana does not mate in a heterothallic manner, its mt A-1 gene functions as a mating activator in N. crassa. In addition, the N. africana mt A-1 gene confers mating type-associated vegetative incompatibility in N. crassa. DNA sequence analysis shows that the N. africana mt A-1 open reading frame (ORF) is 93% identical to that of N. crassa mt A-1. The mt A-1 ORF of N. africana contains no stop codons and was detected as a cDNA which is processed in a similar manner to mt A-1 of N. crassa. By DNA blot and orthogonal field agarose gel electrophoretic analysis, it is shown that the composition and location of the mating-type locus and the organization of the mating-type chromosome of N. africana are similar to that of N. crassa.

Amino Acid Sequence↗
PubMed1991

Molecular characterization of Saccharomyces cerevisiae URA6 gene. DNA sequence, mutagenesis analysis, and cell cycle regulation relevant to its suppression mechanism to cdc8 mutation.

Yeast SOC8 DNA fragment was isolated as a wild type dominant suppressor of cdc8 mutation. We have used Bal31 deletion analysis to define the minimal 1 kilobase HpaI-NcoI DNA element required for complementing the cdc8 mutation. The complementing sequence harbored a multicopy plasmid also enhanced by uridine monophosphate kinase in crude extracts. DNA sequence analysis revealed an open reading frame encoding a protein with a molecular weight of 24,949. Since our SOC8 sequence was identical to that of URA6 gene, which encodes uridine monophosphate kinase, we conclude that SOC8 is allelic with URA6, and we use the term URA6 hereafter. Northern blotting experiments showed that the size of mRNA is about 0.9 kibobases. Primer extension experiments showed multiple transcriptional starting sites primarily located at -160 The size and the deduced amino acid composition are consistent with information obtained from purified uridine monophosphate kinase. Thus, both molecular genetic and biochemical evidence supports a notion that the URA6 is SOC8 encoding a yeast uridine monophosphate kinase. Mutagenesis analysis of its putative nucleotide-binding site, altering essential lysine to glutamic acid, resulted in loss of its uridine monophosphate kinase activity. Complementation analysis studies indicated that the mutated ura6 gene abolished its ability to complement ura6 mutant cells; nor could it suppress cdc8 mutation. Unlike CDC8, the mRNA level of the URA6 gene did not fluctuate throughout the cell cycle; presumably, the temporal order of these two enzymatic activities might be different during cell cycle progression. These data may explain an incomplete suppression of cdc8 by URA6, as previously observed. Taken together, the results support our previous speculation that the suppression of the cdc8 mutation mechanisms by URA6 is due to the provision of the trans-acting dTMP kinase activity to complement the cdc8 defect.

Amino Acid Sequence↗
PubMed1993

Molecular analysis of the hsp (groE) operon of Leptospira interrogans serovar copenhageni.

A chromosomal gene library of Leptospira interrogans serovar copenhageni strain Wijnberg was constructed in phage lambda gt11. Plaque immunoassay with R alpha P64 antiserum identified one clone expressing a putative groEL homologue. DNA sequence analysis of the 2.4 kb EcoRI-Bam HI cloned fragment from strain Wijnberg revealed two open reading frames encoding polypeptides of 10.5 kDa (Hsp10) and 58 kDa (Hsp58). Sequence comparison of the deduced amino acid sequences of these ORFs confirmed the operon as the groE equivalent of Leptospira. Transcriptional analysis suggested that this operon is primarily under the control of an E sigma 70 promoter element. However, both Hsp10 and Hsp58 were overexpressed under heat-shock conditions as determined by [35S]-methionine pulse labelling experiments. As no functional heat-shock promoter could be identified, a 9bp inverted repeat, located between the transcription and translation start sites, may play a role in the upregulation of this operon under heat-shock conditions, similar to mechanisms described for several Gram-positive organisms.

Amino Acid Sequence↗
PubMed1980

Studies of the recognition sequence of phi X174 gene A protein. Cleavage site of phi X gene A protein in St-1 RFI DNA.

It is already known that phi X gene A protein converts besides phi X RFI DNA also the RFI DNAs of the single-stranded bacteriophages G4, St-1, alpha 3 and phi K into RFII DNA. We have extended this observations for bacteriophages G14 and U3. Restriction enzyme analysis placed the phi X gene A protein cleavage site in St-1 RF DNA in the HinfI restriction DNA fragment F10 and in the overlapping HaeIII restriction DNA fragment Z7. The exact position and the nucleotide sequence at the 3'-OH end of the nick were determined by DNA sequence analysis of the single-stranded DNA subfragment of the nicked DNA fragment F10 obtained by gelelectrophoresis in denaturing conditions. A stretch of 85 nucleotides of St-1 DNA around the position of the phi X gene A protein cleavage site was established by DNA sequence analysis of the restriction DNA fragment Z7F1. Comparison of this nucleotide sequence with the previously determined nucleotide sequence around the cleavage site of phi X gene A protein in phi X174 RF DNA and G4 RF DNA revealed an identical sequence of only 10 nucleotides. The results suggest that the recognition sequence of the phi X174 gene A protein lies within these 10 nucleotides.

Bacteriophage phi X 174↗
PubMed2000

Renibacterium salmoninarum isolates from different sources possess two highly conserved copies of the rRNA operon .

The nucleotide sequences of the rRNA genes and the 5' flanking region were determined for R. salmoninarum ATCC 33209T from overlapping products generated by PCR amplification from the genomic DNA. Comparison of the sequences with rRNA genes from a variety of bacteria demonstrated the close relatedness between R. salmoninarum and the high G+C group of the actinobacteria, in particular, Arthrobacter species. A regulatory element within the 5' leader of the rRNA operon was identical to an element, CL2, described for mycobacteria. PCR, DNA sequence analysis, and DNA hybridisation were performed to examine variation between isolates from diverse sources which represented the four 16S-23S rRNA intergenic spacer sequevars previously described for R. salmoninarum. Two 23S-5S rRNA intergenic spacer sequevars of identical length were found. DNA hybridisation using probes complementary to 23S rDNA and 16S rDNA identified two rRNA operons which were identical or nearly identical amongst 40 isolates sourced from a variety of countries.

Actinomycetales↗
PubMed1991

Sequence analysis of DNA from formalin-fixed, paraffin-embedded human malignant melanoma.

Direct sequence analysis of in vitro amplified DNA from paraffin-embedded tissue is a promising new technique useful for detailed molecular analyses of material stored in dermatopathological archives. As only minute amounts of highly damaged DNA can be recovered from paraffin-embedded sections, sequence analysis of DNA from formalin-fixed and embedded tissue can result in less clear and readable data. We report here useful modifications that result in clear and readable sequence data. As an example, we detail the detection of a point mutation at codon 61 of the human N-ras gene in DNA from a formalin-fixed paraffin-embedded malignant melanoma specimen. This technique will be of value in many areas of molecular dermatopathology.

Base Sequence↗
PubMed2001

Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity.

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.

ATP-Binding Cassette Transporters↗
PubMed1998

Isolation and characterization of Staphylococcus aureus starvation-induced, stationary-phase mutants defective in survival or recovery.

Ten Staphylococcus aureus mutants, defective in the starvation-induced stationary phase of growth were isolated from two independent Tn917-LTV1 transposon insertion libraries and were designated suv as they had apparent survival defects. Seven of these mutants were defective under amino-acid-limiting conditions alone. Two mutants (suv-3 and suv-20) demonstrated lower plating efficiency when starved for glucose, phosphate or amino acids and one mutant (suv-11) had reduced plating efficiency after amino acid or glucose starvation. All of the mutants tested were as resistant to hydrogen peroxide assault as the parent, but six were more sensitive to low pH conditions. All the mutants were physically mapped on the S. aureus chromosome using PFGE. Chromosomal DNA flanking the Tn917-LTV1 insertion sites was rescued by cloning into Escherichia coli. DNA sequence analysis resulted in the identification of a number of transposon-disrupted ORFs encoding putative components such as superoxide dismutase (suv-1), haem A synthase (suv-3), a component of the SOS response (suv-9) and hypoxanthine-guanine phosphoribosyltransferase (suv-20). The Tn917-LTV1 insertion created lacZ transcriptional fusions for some of the stationary-phase loci. Expression analysis indicated that suv-4 was induced at mid-exponential phase, whereas suv-3 and suv-11 were induced at the onset of stationary phase. The possible roles of these suv components in stationary-phase survival or recovery is discussed.

DNA Transposable Elements↗