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PubMed1993

Refined physical mapping of chromosome 16-specific low-abundance repetitive DNA sequences.

Repetitive DNA sequences have been implicated in the origin of several disease phenotypes, including fragile X syndrome, myotonic dystrophy, and spinal bulbar atrophy. In addition, a complex family of chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been mapped by fluorescence in situ hybridization to regions of chromosome 16 that undergo breakage/rearrangement in acute nonlymphocytic leukemia (ANLL) cells. It has been hypothesized that these repetitive sequences are causally related to the chromosome rearrangements found in ANLL. Here, we further refine the mapping of CH16LAR sequences with respect to the ANLL inversion breakpoints, using a panel of somatic cell hybrids containing 51 different chromosome 16 breakpoints. These studies indicate that CH16LAR sequences at 16p13 are in close proximity to the ANLL short-arm breakpoint region. However, the region containing the highest density of CH16LAR sequences on the long arm appears to be distal to the region where the ANLL long-arm breakpoint has been mapped. These studies further show that CH16LAR sequences map in close proximity to FRA16D and FRA16A.

Animals↗
PubMed2006

Effectiveness of MR imaging for diagnosing the mild forms of acute pancreatitis: comparison with MDCT.

PURPOSE: To assess the usefulness of MRI and determine which MR sequence is most effective for diagnosing the mild forms of acute pancreatitis. MATERIALS AND METHODS: Forty subjects (20 normal volunteers and 20 patients with a mild form of acute pancreatitis) underwent MRI with fat-suppressed T1-weighted fast low-angle shot (FLASH), half-Fourier acquisition single-shot turbo spin-echo (HASTE), and TSE short TI inversion recovery (TSE-STIR) sequences. The 20 patients with a mild form of acute pancreatitis underwent multidetector CT (MDCT) and MRI within a 24-hour interval. We qualitatively analyzed all of the images by assessing inflammatory changes in the pancreas and peripancreatic fat. We quantitatively compared differences in pancreas-liver contrast between the control and patient groups for each MR sequence by measuring the signal intensities of the pancreas and liver. RESULTS: TSE-STIR was the best of the four modalities for delineating peripancreatic and pancreatic inflammation (P < 0.01). TSE-STIR depicted definitive peripancreatic and pancreatic inflammation in 18 and 15 patients, respectively. MDCT depicted only three cases of peripancreatic inflammation. TSE-STIR was also produced the best the best quantitative results of the MR sequences (P = 0.09). CONCLUSION: MRI is helpful for diagnosing the mild forms of acute pancreatitis. We recommend the use of TSE-STIR imaging as part of the routine protocol for evaluating pancreatitis.

Adult↗
PubMed2002

Identification and analysis of the amylase-binding protein B (AbpB) and gene (abpB) from Streptococcus gordonii.

The binding of salivary amylase to Streptococcus gordonii has previously been shown to involve a 20-kDa amylase-binding protein (AbpA). S. gordonii also releases an 82-kDa protein into the supernatant that binds amylase. To study this 82-kDa component, proteins were precipitated from bacterial culture supernatants by the addition of acetone or purified amylase. Precipitated proteins were separated by SDS-PAGE and transferred to a sequencing membrane. The P2 kDa band was then sequenced, yielding a 25 N-terminal amino acid sequence, CGFIFGRQLTADGSTMFGPTEDYP. Primers derived from this sequence were used in an inverse PCR strategy to clone the full-length gene from S. gordonii chromosomal DNA. An open reading frame of 1959 bp was noted that encoded a 652 amino acid protein having a predicted molecular mass of 80 kDa. The first 24 amino acid residues were consistent with a hydrophobic signal peptide, followed by a 25 amino acid N-terminal sequence that shared identity (24 of 25 residues) with the amino acid sequence of purified AbpB. The abpB gene from strains of S. gordonii was interrupted by allelic exchange with a 420-bp fragment of the abpB gene linked to an erythromycin cassette. The 82-kDa protein was not detected in supernatants from these mutants. These abpB mutants retained the ability to bind soluble amylase. Thus, AbpA, but not AbpB, appears sufficient to be the major receptor for amylase binding to the streptococcal surface. The role of AbpB in bacterial colonization remains to be elucidated.

Amino Acid Sequence↗
PubMed1999

Mechanism and application of genetic recombination in herpesviruses.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous human pathogen that latently infects sensory ganglia and encodes over 80 genes in a 152 kbp DNA genome. This well characterised virus provides a model for analysing genetic recombination in herpesviruses, a fundamental biological process by which new combinations of genetic materials are generated. The frequency of homologous recombination was estimated to be 0.0048-0.007 (0.48%-0.7%)/kb of the HSV-1 genome, determined using physical markers. The double-strand break repair model, the current model of homologous recombination, adequately explains L-S inversion of herpesvirus genomes and the recombinogenicity of the a sequence. Several herpesvirus genomes, including HSV-1 consist of a unique sequence bracketed by a pair of inverted repeat sequences. This arrangement is attributed to illegitimate recombination between molecules arranged in an inverse orientation. Junctions of unique and repeated sequences that correspond to the crossover site of illegitimate recombination are recombinogenic. Recombination is important for virus evolution, construction of mutated virus, gene therapy and vaccination in which the potential for recombination between engineered input virus and wild type virus has to be considered.

Animals↗
PubMed1998

Unselected brain 0.5-T MR imaging: comparison of lesion detection and characterization with three T2-weighted sequences.

PURPOSE: To compare the diagnostic values of three T2-weighted sequences in unselected brain 0.5-T magnetic resonance (MR) imaging examinations. MATERIALS AND METHODS: MR imaging was performed in 102 patients because of neurologic symptoms or the follow up "previously known disease." The protocol comprised three T2-weighted sequences: turbo spin-echo (SE), turbo fluid-attenuated inversion-recovery (FLAIR), and gradient and SE (GRASE) sequences. In a prospective, blinded analysis, sequences were compared quantitatively and qualitative by three independent readers for lesion detection and characterization. RESULTS: Twenty-four patients had normal findings, and 78 had abnormal findings, with 121 final diagnoses. Quantitatively, the "white" cerebrospinal fluid (CSF) sequences, turbo SE and GRASE, had higher contrast-to-noise ratios (CNRs) than the "black" CSF sequence, FLAIR, with respect to the signal intensity of white manner, but lower CNRs than FLAIR with respect to the signal intensity of CSF. Qualitatively, for lesion detection, sensitivity and accuracy were significantly higher for FLAIR than for turbo SE or GRASE (P < .05); there was no statistically significant difference between turbo SE or GRASE. FOR lesion characterization, FLAIR was significantly superior to GRASE and turbo SE for white matter disease (P < .05), superior to only turbo SE (P < .05) for vascular disease, and not superior to GRASE or turbo SE for tumors. CONCLUSIONS: The black CSF T2-weighted turbo FLAIR sequence is a useful adjunct to the usual white CSF T2-weighted sequences in unselected brain 0.5-T MR imaging.

Adolescent↗
PubMed1989

Imaging of pancreatic neoplasms: comparison of MR and CT.

Thirty-two patients with pathologically proved pancreatic carcinomas or cystadenomas were evaluated with MR images obtained with T1-weighted spin echo (short TR/short TE), inversion recovery, and T2-weighted spin-echo (long TR/long TE) pulse sequences. CT was used as the reference standard to determine the ability of MR to delineate normal and abnormal pancreatic anatomy and thereby to exclude or detect pancreatic malignancy. Short TR/short TE spin-echo sequences were significantly better (p less than .05) than inversion recovery or T2-weighted spin-echo sequences in resolution of both normal and abnormal anatomy. Resolution of pancreatic anatomy correlated (r = .9) with the image signal-to-noise ratio. In seven (22%) of 32 cases, MR visualized pancreatic tumors better than CT did because it showed a signal intensity difference between the tumor and normal pancreatic tissue. Overall, the slight superiority of MR over CT for tumor visualization tended to occur in larger tumors and was not statistically significant. On T1-weighted images, 63% (20 of 32) of pancreatic tumors studied had lower signal intensities than normal pancreatic tissue, whereas on T2-weighted sequences (TE = 60, 120, and 180 msec) only 41% (13 of 32) of tumors had increased signal intensities. Currently available MR imaging techniques offer no significant advantages over CT for evaluating the pancreas for neoplasia.

Adult↗
PubMed1989

Pseudogenes and short repeated sequences in the rice chloroplast genome.

The rice chloroplast genome has been derived from a tobacco-like ancestral form by three major inversions. In the rice genome we have found six pseudogenes, psi trnG, psi trnI, psi 3'-rps 12a, psi trnT, psi trnE and psi trnfM/G, all located near inversion endpoints, as well as four short repeated sequences. A comparison of rice, wheat and tobacco sequences indicated that similar pseudogenes are present in wheat but not in tobacco, suggesting that the creation of these pseudogenes occurred before the divergence of rice and wheat. The region downstream of rbcL is a variable region and contains psi rpl23 in rice and wheat and another psi 3'-rps 12b further downstream in rice. This psi 3'-rps 12b shows a higher homology to the functional rps 12 than psi 3'-rps 12a, which suggests that it appeared more recently. The involvement of these pseudogenes in genome inversions and the creation of the pseudogenes and short repeated sequences are discussed.

Base Sequence↗
PubMed2000

Retro-inverso prosaptide peptides retain bioactivity, are stable In vivo, and are blood-brain barrier permeable.

Prosaptide (trademark of Myelos Corporation, San Diego, CA) peptides are based on the 14-amino-acid neurotrophic sequence of human prosaposin and, like the parent protein, have potent neurotrophic and neuroprotective properties. We previously examined the in vivo stability of a series of bioactive Prosaptide peptides and designed peptides with increased enzymatic stability in the central and peripheral nervous systems. In this article, we examined the stability, biological activity, and permeability of the blood-brain barrier to retro-inverso Prosaptide peptidomimetics. Retro-inversion both reverses the primary sequence and replaces L-amino acids with D-amino acids. We examined the bioactivity of five peptidomimetics, Prosaptides D1-D5. Prosaptide D1, a peptide containing all D-amino acids with the primary sequence intact, was inactive. However, four retro-inverso peptidomimetics, Prosaptides D2-D5 retained bioactivity in neurite outgrowth and [(35)S]GTPgammaS binding assays. We focused on Prosaptide D4 as a prototypical retro-inverso Prosaptide peptidomimetic for further study. (125)I-Prosaptide D4 remained intact in brain or serum for 60 min after i.v. administration and was transported across the blood-brain barrier with a unidirectional influx constant of 2.5 x 10(-4) ml. g(-1). min(-1). We conclude that retro-inverso Prosaptide peptidomimetics are excellent candidates for development as therapeutics for central nervous system neurodegeneration.

Amino Acid Sequence↗
PubMed1993

Transposon-induced inversion in Antirrhinum modifies nivea gene expression to give a novel flower color pattern under the control of cycloidearadialis.

The nivea (niv) gene of Antirrhinum majus encodes chalcone synthase, an enzyme involved in synthesis of anthocyanin pigments. The nivrec:98 allele contains a single copy of the transposon Tam3 inserted at the niv locus. A large chromosomal rearrangement derived from this mutant has been shown to be flanked by two copies of Tam3. In this study, we compared sequences involved in this rearrangement with their progenitor sequences and concluded that the rearrangement is an inversion resulting from an aberrant transposition occurring shortly after replication of Tam3 that left both copies of Tam3 active after the rearrangement. Excision of Tam3 from its position adjacent to the niv coding region resulted in a novel distribution of anthocyanin pigment in the flower tube, caused by the interaction of the new sequences with the remnant of the niv promoter. The new sequences upstream of niv serve both to enhance niv transcription and to redirect the pattern of gene expression, placing niv under the control of the gene cycloidearadialis, which determines the morphogenetic polarity of the flower.

Alleles↗
PubMed2000

Thermal lesion conspicuity following interstitial radiofrequency thermal tumor ablation in humans: a comparison of STIR, turbo spin-echo T2-weighted, and contrast-enhanced T1-weighted MR images at 0.2 T.

The purpose of this study was to compare the contrast between radiofrequency (RF) thermal liver lesions and surrounding tissue in T2-weighted turbo spin-echo sequences (TSE T2), short TI inversion recovery techniques (STIR), and contrast-enhanced (CE) T1-weighted spin-echo images. Nineteen RF thermal ablations were performed on eight patients with metastatic liver tumors. After ablation, contrast-to-noise ratios (CNRs) were calculated between mean signal amplitudes from three regions of interest (ROI) (lesion, surrounding edema, and normal tissue) using TSE T2-weighted, STIR, and contrast-enhanced T1-weighted (CE T1) sequences for each lesion. CNRs between the thermal lesion and normal liver tissue for both TSE T2-weighted (mean 0.9) and STIR (2.0) images were significantly lower than for CE T1-weighted (8.4) images (t-test, alpha = 0.05). However, CNRs between edema rim and the core of the thermal lesion for both TSE T2-weighted (8.1) and STIR images (7.2) were not significantly different (t-test, alpha = 0.05) from CNRs between lesion and normal tissue for CE T1-weighted images (8.4), nor was the CNR between edema rim and normal tissue for both TSE T2-weighted (10.3) and STIR (9.8) images. Although the edema was not visible on CE T1-weighted images, 18 of 19 lesions (94.7%) were surrounded by a hyperintense rim on TSE T2-weighted or STIR images. Both TSE T2-weighted and STIR sequences represent valid techniques for repeatable assessment of RF thermal lesions.

Adult↗
PubMed1994

A reliable amplification technique with single-sided specificity for the isolation of 5' gene-regulating regions.

A simple and efficient method is described for the isolation of extension fragments of known DNA sequences by polymerase chain reaction (PCR) using a single specific primer. With this method, size-selected genomic DNA fragments are ligated to a plasmid vector (pGEM-4Z) which contains sequencing primers and the population of chimeric plasmids is used for transforming Escherichia coli. DNA is extracted from an aliquot of the resulting mini-library and PCR performed using a sequence-specific primer and either of the standard sequencing primers of the plasmid vector. This method appears to be more versatile than inverse PCR (IPCR), since: (i) the DNA sequence needed as the specific primer can be as short as about 20 nucleotides (nt); (ii) the DNA templates to be used in PCR are available in high amount, thus facilitating all manipulations; and (iii) if relinearization of the DNA by restriction enzyme digestion is desired before the PCR reaction, many restriction sites can be chosen from the vector polylinker. Using this method, we have isolated the genomic 5' region of the carrot bifunctional dihydrofolate reductase-thymidylate synthase-encoding gene dhfr-ts using a 21-nt sequence of the 5' region of the dhfr-ts cDNA clone as the specific primer.

Base Sequence↗
PubMed2003

[Comparison of three-dimensional gradient echo, turbo spin echo and steady-state gradient echo sequences in axial MRI examination of the cervical spine].

PURPOSE: The aim of this study was to assess the detectability of the structures that are affected by cervical spondylosis by different 3D MRI sequences and compare the image quality of those sequences. MATERIALS AND METHODS: Twenty healthy volunteers were examined using a 1.5 T MR unit. T2* weighted 3D-gradient echo sequence with magnetization transfer saturation pulse (3D-FFE-MTC), T2 weighted 3D turbo spin echo (TSE) sequence with and without spectral presaturation inversion recovery pulse (T2-3D-TSE/SPIR+ and T2-3D-TSE/SPIR-) and balanced-FFE (b-FFE) sequences were compared. Spinal cord signal-to-noise ratio (SNR), cerebrospinal fluid (CSF) SNR, and CSF-spinal cord contrast-to-noise ratio (CNR) were calculated quantitatively. The detectability of neural foramina, spinal nerve roots, uncinate process and ligamentum flava were graded on a 5-point scale (0: minimum, 4: maximum) qualitatively. The presence of the artifacts and overall image quality were graded on a 4-point scale (0: minimum, 3: maximum). RESULTS: Balanced-FFE sequence yielded the best results for each of three quantitative evaluation. In terms of qualitative evaluation, for the uncinate process T2-3D-TSE/SPIR- sequence was superior to the other three sequences (p < 0.01). For the ligamentum flava T2-3D-TSE/SPIR- and b-FFE sequences were superior to the other two (p < 0.01). For the neural foramina b-FFE yielded the lowest score (3), however the other three sequences were not significantly different (3.2-3.5) (p > 0.05). Artifacts were most commonly seen using 3D-FFE-MTC. In terms of overall image quality T2-3D-TSE/SPIR- yielded the highest score, followed by b-FFE. CONCLUSION: 3D-FFE-MTC sequence is frequently preferred for cervical spinal MRI studies. However our study yielded best scores for T2-3D-TSE/SPIR, followed by b-FFE in the quantitative and qualitative evaluation of the structures affected by cervical spondylosis.

Adolescent↗
PubMed1992

Structural analysis of a carcinogen-induced genomic rearrangement event.

We have explored the mechanism of genomic rearrangement in a hamster fibroblast cell culture system in which rearrangements are induced 5' to the endogenous thymidine kinase gene by chemical carcinogen treatment. The wild-type region around one rearrangement breakpoint was cloned and sequenced. With this sequence information, the carcinogen-induced rearrangement was cloned from the corresponding rearranged cell line by the inverse polymerase chain reaction. After the breakpoint fragment was sequenced, the wild-type rearrangement partner (RP15) was isolated by a second inverse polymerase chain reaction of unrearranged DNA. Comparison of the sequence of the rearrangement breakpoint with the wild-type RP15 and 5' thymidine kinase gene regions revealed short repeats directly at the breakpoint, as well as nearby A + T-rich regions in each rearrangement partner. Pulsed-field electrophoresis analysis demonstrated that this rearrangement is an interstitial deletion of 35 kilobases. Southern blot analysis of the RP15 region in unrearranged parental cells showed a demethylated CpG island and a complex of DNase I-hypersensitive sites adjacent to the breakpoint in the region deleted by the rearrangement. Therefore, these studies reveal interesting sequence and chromatin features near the rearrangement breakpoints and suggest a role for nuclear organization in the mechanism of carcinogen-induced genomic rearrangement.

Animals↗
PubMed2006

Assessment of myocardial viability using delayed enhancement magnetic resonance imaging at 3.0 Tesla.

OBJECTIVE: Cardiac magnetic resonance imaging (MRI) at 3.0 T has recently become available and potentially provides a significant improvement of tissue contrast in T1-weighted imaging techniques relying on Gd-based contrast enhancement. Imaging at high-field strength may be especially advantageous for methods relying on strong T1-weighting and imaging after contrast material administration. The aim of this study was to compare cardiac delayed enhancement (DE) MRI at 3.0 T and 1.5 T with respect to image quality, signal-to-noise ratio (SNR), and contrast-to-noise ratio (CNR) between infarcted and normal myocardium. MATERIALS AND METHODS: Forty consecutive patients with history of myocardial infarction were examined at 3.0 T (n = 20) or at 1.5 T (n = 20). Myocardial function was assessed using cine steady-state-free-precession (SSFP) sequences (TR 3.1 milliseconds, TE 1.6 milliseconds, flip angle 70 degrees , and a matrix of 168 x 256 at 1.5 T and TR 3.4 milliseconds, TE 1.7 milliseconds, flip angle 50 degrees and a matrix of 168 x 256 at 3.0 T), acquired in long- and short-axes views. DE images were obtained 15 minutes after the administration of 0.15 mmol of Gd-DTPA/kg body weight using a segmented inversion recovery prepared gradient echo sequence at 1.5 T (TR 9.6 milliseconds, TE 4.4 milliseconds, flip angle 25 degrees , matrix 160 x 256, bandwidth 140 Hertz/pixel) and at 3.0 T (TR 9.8 milliseconds, TE 4.3 milliseconds, flip angle 30 degrees , matrix 150 x 256, bandwidth 140 Hertz/pixel). For image analysis, standardized SNR and CNR measurements were performed in infarcted and remote myocardial regions. Two independent observers rated image quality on a 4-point scale (0 = poor image quality, 1 = sufficient image quality, 2 = good image quality, 3 = excellent image quality). RESULTS: High diagnostic image quality was obtained in all patients. Rating of mean image quality was 2.2 +/- 0.8 at 1.5 T and 2.5 +/- 0.6 at 3.0 T (P = 0.012) for observer 1 and 2.2 +/- 0.7 at 1.5 T and 2.6 +/- 0.6 at 3.0 T (P = 0.003) for observer 2, respectively. Interobserver agreement was good (kappa = 0.68 at 1.5 T and 0.78 at 3.0 T). SNR measurements yielded a mean SNR of 37.8 +/- 13.9/22.9 +/- 6.0 in infarcted myocardium (P < 0.001) and 5.6 +/- 2.2/5.9 +/- 2.4 in normal myocardium (P = 0.45) at 3.0 T/1.5 T, respectively. CNR measurements revealed mean values of 32.4 +/- 13.0/16.7 +/- 5.4 (P< 0.001) at 3.0 T/1.5 T, respectively. CONCLUSIONS: Delayed enhancement MRI at 3.0 T is feasible and provides superior image quality compared with 1.5 T. Furthermore, using identical contrast doses, increased SNR and CNR values were recorded at 3.0 T.

Adult↗
PubMed2002

The evolution of DNA regulatory regions for proteo-gamma bacteria by interspecies comparisons.

The comparison of homologous noncoding DNA for organisms a suitable evolutionary distance apart is a powerful tool for the identification of cis regulatory elements for transcription and translation and for the study of how they assemble into functional modules. We have fit the three parameters of an affine global probabilistic alignment algorithm to establish the background mutation rate of noncoding sequence between E. coli and a series of gamma proteobacteria ranging from Salmonella to Vibrio. The lower bound we find to the neutral mutation rate is sufficiently high, even for Salmonella, that most of the conservation of noncoding sequence is indicative of selective pressures rather than of insufficient time to evolve. We then use a local version of the alignment algorithm combined with our inferred background mutation rate to assign a significance to the degree of local sequence conservation between orthologous genes, and thereby deduce a probability profile for the upstream regulatory region of all E. coli protein-coding genes. We recover 75%-85% (depending on significance level) of all regulatory sites from a standard compilation for E. coli, and 66%-85% of sigma sites. We also trace the evolution of known regulatory sites and the groups associated with a given transcription factor. Furthermore, we find that approximately one-third of paralogous gene pairs in E. coli have a significant degree of correlation in their regulatory sequence. Finally, we demonstrate an inverse correlation between the rate of evolution of transcription factors and the number of genes they regulate. Our predictions are available at http://www.physics.rockefeller.edu/([tilde-see text])siggia.

Algorithms↗
PubMed2006

INFO-RNA--a fast approach to inverse RNA folding.

MOTIVATION: The structure of RNA molecules is often crucial for their function. Therefore, secondary structure prediction has gained much interest. Here, we consider the inverse RNA folding problem, which means designing RNA sequences that fold into a given structure. RESULTS: We introduce a new algorithm for the inverse folding problem (INFO-RNA) that consists of two parts; a dynamic programming method for good initial sequences and a following improved stochastic local search that uses an effective neighbor selection method. During the initialization, we design a sequence that among all sequences adopts the given structure with the lowest possible energy. For the selection of neighbors during the search, we use a kind of look-ahead of one selection step applying an additional energy-based criterion. Afterwards, the pre-ordered neighbors are tested using the actual optimization criterion of minimizing the structure distance between the target structure and the mfe structure of the considered neighbor. We compared our algorithm to RNAinverse and RNA-SSD for artificial and biological test sets. Using INFO-RNA, we performed better than RNAinverse and in most cases, we gained better results than RNA-SSD, the probably best inverse RNA folding tool on the market. AVAILABILITY: www.bioinf.uni-freiburg.de?Subpages/software.html.

Algorithms↗
PubMed2000

Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region.

We have constructed an integrated cytogenetic map of chromosome arm 4S of Arabidopsis thaliana. The map shows the detailed positions of various multicopy and unique sequences relative to euchromatin and heterochromatin segments. A quantitative analysis of the map positions at subsequent meiotic stages revealed a striking pattern of spatial and temporal variation in chromatin condensation for euchromatin and heterochromatin. For example, the centromere region consists of three domains with distinguishable structural, molecular, and functional properties. We also characterized a conspicuous heterochromatic knob of approximately 700 kb that accommodates a tandem repeat and several dispersed pericentromere-specific repeats. Moreover, our data provide evidence for an inversion event that relocated pericentromeric sequences to an interstitial position, resulting in the heterochromatic knob.

Arabidopsis↗
PubMed1999

Random transposition by Tn916 in Desulfitobacterium dehalogenans allows for isolation and characterization of halorespiration-deficient mutants.

To allow for the molecular analysis of halorespiration by the strictly anaerobic gram-positive bacterium Desulfitobacterium dehalogenans, halorespiration-deficient mutants were selected and characterized following insertional mutagenesis by the conjugative transposon Tn916. To facilitate rapid screening of transconjugants, a highly efficient method for the growth of single colonies on solidified medium has been developed. A streptomycin-resistant mutant of D. dehalogenans was isolated and mated with Enterococcus faecalis JH2-2 carrying Tn916. Insertion of one or two copies of Tn916 into the chromosome of D. dehalogenans was observed. From a total of 2,500 transconjugants, 24 halorespiration-deficient mutants were selected based upon their inability to use 3-chloro-4-hydroxyphenylacetic acid as an electron acceptor. Physiological characterization led to the definition of three phenotypic classes of mutants that differed in their ability to use the additional terminal electron acceptors nitrate and fumarate. The activities of hydrogenase and formate dehydrogenase were determined, and the transposon insertion sites in selected mutants representing the different classes were analyzed on the sequence level following amplification by inverse PCR. The results of the molecular characterization as well as the pleiotropic phenotypes of most mutants indicate that genes coding for common elements shared by the different respiratory chains present in the versatile D. dehalogenans have been disrupted.

Bacteria, Anaerobic↗