scholarly journals Characterization of the Plasmid Encoded Virulence Region pat-1 of Phytopathogenic Clavibacter michiganensis subsp. michiganensis

1997 ◽  
Vol 10 (2) ◽  
pp. 195-206 ◽  
Author(s):  
Jens Dreier ◽  
Dietmar Meletzus ◽  
Rudolf Eichenlaub
Keyword(s):  
Structural Gene ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Transcription Initiation ◽  
Serine Proteases ◽  
Tandem Repeats ◽  
Sequence Motif ◽  
Clavibacter Michiganensis ◽  
Reading Frame ◽  
Clavibacter Michiganensis Subsp

The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, causing bacterial wilt and canker, harbors two plasmids, pCM1 (27.5 kb) and pCM2 (72 kb), carrying genes involved in virulence. The region of plasmid pCM2 encoding the pathogenicity locus pat-1 was mapped by deletion analysis and complementation studies to a 1.5-kb BglII/SmaI DNA fragment. Introduction of the pat-1 region into endophytic, plasmid-free isolates of C. michiganensis subsp. michiganensis converted these bacteria into virulent pathogens. Based on the nucleotide sequence of the pat-1 region, an open reading frame (ORF1) can be predicted, coding for a protein of 280 amino acids and 29.7 kDa with homology to serine proteases. Introduction of a frame-shift mutation in ORF1 leads to a loss of the pathogenic phenotype. Northern (RNA) hybridizations identified an 1.5-knt transcript of the pat-1 structural gene. The site of transcription initiation was mapped by primer extension and a typical -10/-35 region was located with significant homology to the consensus Escherichia coli σ70 and Bacillus subtilis σ43 promoters. Downstream of the pat-1 structural gene, a peculiar repetitive sequence motif (pat-1rep) is located, consisting of 20 direct tandem repeats preceded by a run of 14 guanosine residues. DNA sequences homologous to pat-1rep were isolated and characterized from four virulent C. michiganensis subsp. mich-iganensis strains exhibiting a high extent of structural conservation. The deletion of this repetitive sequence reduced virulence significantly but did not lead to a complete loss of the virulence phenotype.

scholarly journals Specific Detection of Clavibacter michiganensis subsp. sepedonicus by Amplification of Three Unique DNA Sequences Isolated by Subtraction Hybridization

1997 ◽  
Vol 87 (8) ◽  
pp. 853-861 ◽  
Author(s):  
Dallice Mills ◽  
Brian W. Russell ◽  
Janet Williams Hanus
Keyword(s):  
Dna Sequences ◽  
Bacterial Species ◽  
Single Copy ◽  
Cross Reactivity ◽  
Clavibacter Michiganensis ◽  
Subtraction Hybridization ◽  
Detection Systems ◽  
Clavibacter Michiganensis Subsp ◽  
Polymerase Chain ◽  
Primer Sets

Three single-copy, unique DNA fragments, designated Cms50, Cms72, and Cms85, were isolated from strain CS3 of Clavibacter michiganensis subsp. sepedonicus by subtraction hybridization using driver DNA from C. michiganensis subsp. insidiosus, C. michiganensis subsp. michiganensis, and Rhodococcus facians. Radio-labeled probes made of these fragments and used in Southern blot analysis revealed each to be absolutely specific to all North American C. michiganensis subsp. sepedonicus strains tested, including plasmidless and nonmucoid strains. The probes have no homology with genomic DNA from related C. michiganensis subspecies insidiosus, michiganensis, and tessellarius, nor with DNA from 11 additional bacterial species and three unidentified strains, some of which have been previously reported to display cross-reactivity with C. michiganensis subsp. sepedonicus-specific antisera. The three fragments shared no homology, and they appeared to be separated from each other by at least 20 kbp in the CS3 genome. Internal primer sets permitted amplification of each fragment by the polymerase chain reaction (PCR) only from C. michiganensis subsp. sepedonicus DNA. In a PCR-based sensitivity assay using a primer set that amplifies Cms85, the lowest level of detection of C. michiganensis subsp. sepedonicus was 100 CFU per milliliter when cells were added to potato core fluid. Erroneous results that may arise from PCR artifacts and mutational events are, therefore, minimized by the redundancy of the primer sets, and the products should be verifiable with unique capture probes in sequence-based detection systems.

Expression of tissue-specific Ren-1 and Ren-2 genes of mice: comparative analysis of 5'-proximal flanking regions

1984 ◽  
Vol 4 (11) ◽  
pp. 2321-2331
Author(s):  
L J Field ◽  
W M Philbrick ◽  
P N Howles ◽  
D P Dickinson ◽  
R A McGowan ◽  
...  
Keyword(s):  
Structural Gene ◽  
Transcription Initiation ◽  
Submaxillary Gland ◽  
Inbred Strains ◽  
Initiation Site ◽  
Sequence Motifs ◽  
Specific Expression ◽  
Tissue Specific ◽  
Reading Frame ◽  
A Minor

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed submaxillary gland renin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue-specific expression. In an effort to understand the molecular basis for this differential expression, detailed analysis of the genomic sequences corresponding to the Ren-1 and Ren-2 genes, and the transcripts originating from these loci, was undertaken. Sequence analysis of regions proximal to the structural genes indicated the presence of eucaryotic consensus sequences for transcription. These sequence motifs were strongly conserved between Ren-1 and Ren-2. Approximately 150 bases upstream from the major transcription initiation site, significant differences between these genes were apparent, including the presence of a repetitive DNA element in the Ren-2 copy as well as other breaks in homology and sequence curiosities. Strong homology between Ren-1 and Ren-2 resumed at a point ca. 200 bases further upstream on Ren-1. S1 analysis of submaxillary gland and kidney RNA populations indicated that the majority of transcripts initiate at homologous positions on Ren-1 and Ren-2. On a per cell basis, the accumulation of Ren-1 transcripts in the kidney and Ren-2 transcripts in the submaxillary gland are probably equivalent. These results suggest that it is tissue-specific utilization of the homologous start sites that is critical to their differential patterns of expression. Models which can account for this observation are presented. Interestingly, we found a minor fraction of transcripts initiating 5' to the major transcription start site. These transcripts encoded an open reading frame which may add an additional 23 amino acids to the N-terminus of the renin precursor.

scholarly journals Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide

1994 ◽  
Vol 299 (2) ◽  
pp. 381-387 ◽  
Author(s):  
G W Black ◽  
G P Hazlewood ◽  
G P Xue ◽  
C G Orpin ◽  
H J Gilbert
Keyword(s):  
Dna Sequences ◽  
Tandem Repeats ◽  
Northern Blot Analysis ◽  
Catalytic Domain ◽  
Catalytic Properties ◽  
Single Gene ◽  
Reading Frame ◽  
Catalytic Domains ◽  
Neocallimastix Patriciarum ◽  
Derivatives Of

A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N. patriciarum xynA cDNA encoding xylanase A. A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated. Northern-blot analysis of mRNA from Avicel-grown N. patriciarum showed that xynB hybridized to a 3.4 kb mRNA species. The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066. The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases. The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times. Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain. To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography. The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose. The XYLB fusion did not cleave any cellulosic substrates. The data presented in this report suggest that the multiple xylanases of N. patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus. The possible origin of the xynB gene is discussed.

scholarly journals Structure and expression of the human immunoglobulin lambda genes.

1990 ◽  
Vol 172 (2) ◽  
pp. 609-620 ◽  
Author(s):  
T J Vasicek ◽  
P Leder
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Rna Splicing ◽  
Transcription Initiation ◽  
Initiation Site ◽  
Enhancer Activity ◽  
Initiation Point ◽  
Reading Frame ◽  
Duplication Events

We determined the DNA sequence of two large regions of chromosome 22: 33.7 kb containing the C lambda complex; and 5.2 kb 5' of the functionally rearranged lambda gene from the human myeloma, U266. Analysis of these sequences reveals the complete structure of the human C lambda complex and a previously undescribed seventh C lambda region that may encode the Ke+Oz- lambda protein. The seven constant regions are organized in a tandem array, and each is preceded by a single J lambda region. lambda 1, lambda 2, lambda 3, and lambda 7 are apparently active genes, while lambda 4, lambda 5, and lambda 6 are pseudogenes. There are no other J lambda or C lambda regions within a 60-kb region surrounding the C lambda complex; however, there are at least four other lambda-like genes and lambda pseudogenes in the human genome. The lambda genes appear to have evolved via a series of gene duplication events resulting from unequal crossing over or gene conversion between the highly conserved C lambda regions on mispaired chromosomes. The lack of Alu sequences in this large segment of DNA suggests that the C lambda complex resulted from a recent amplification of a smaller Alu-free segment of DNA. Illegitimate recombination between repeated sequences containing lambda 2 and lambda 3 may be responsible for variable amplification of the lambda genes. We also found a 1,377-bp open reading frame (ORF) located on the opposite strand in the region containing lambda 7. While this ORF is flanked by potential RNA splicing signals, we have no evidence that it is part of a functional gene. We also discovered a V lambda pseudogene, called psi V lambda 1, 3 kb upstream of the U266 lambda gene. Using primer extension analysis to map the transcription start in the human lambda gene, we have identified its initiation point 41 bp upstream of the initiation codon. Analysis of the lambda promoter reveals that it contains a TATAA box at position -29 relative to the transcription initiation site and an octamer sequence at -67. Computer analysis of 40 kb of DNA sequences surrounding the human lambda locus has revealed no sequences resembling the kappa or IgH transcriptional enhancers, nor have in vitro analyses for function revealed enhancer activity. A comparison of these results with those obtained in separate studies with transgenic mice point to a complex, developmentally linked mechanism of transcriptional activation.

scholarly journals Control of Transcription Initiation by Biased Thermal Fluctuations on Repetitive Genomic Sequences

2020 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Yuji Tokunaga ◽  
Ariel Afek ◽  
Hiroki Takahashi ◽  
Nobuo Shimamoto ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Dna Sequences ◽  
Repetitive Sequence ◽  
Transcription Initiation ◽  
High Throughput Sequencing ◽  
Thermal Fluctuations ◽  
A Genome ◽  
Sequence Elements ◽  
Promoter Dna

In the process of transcription initiation by RNA polymerase, promoter DNA sequences affect multiple reaction pathways determining the productivity of transcription. However, the question of how the molecular mechanism of transcription initiation depends on sequence properties of promoter DNA remains poorly understood. Here, combining the statistical mechanical approach with high-throughput sequencing results, we characterize abortive transcription and pausing during transcription initiation by Escherichia coli RNA polymerase at a genome-wide level. Our results suggest that initially transcribed sequences enriched with thymine bases represent the signal inducing abortive transcription. On the other hand, certain repetitive sequence elements broadly embedded in promoter regions constitute the signal inducing pausing. Both signals decrease the productivity of transcription initiation. Based on solution NMR and in vitro transcription measurements, we also suggest that repetitive sequence elements of promoter DNA modulate the rigidity of its double-stranded form, which profoundly influences the reaction coordinates of the productive initiation via pausing.

scholarly journals Comparative Genomic Analyses of Clavibacter michiganensis subsp. insidiosus and Pathogenicity on Medicago truncatula

2018 ◽  
Vol 108 (2) ◽  
pp. 172-185 ◽  
Author(s):  
You Lu ◽  
Carol A. Ishimaru ◽  
Jane Glazebrook ◽  
Deborah A. Samac
Keyword(s):  
Medicago Truncatula ◽  
Serine Proteases ◽  
Plant Pathogens ◽  
Blue Pigment ◽  
Comparative Genomic ◽  
Clavibacter Michiganensis ◽  
Genome Sequences ◽  
Gram Positive ◽  
Model Legume ◽  
Clavibacter Michiganensis Subsp

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen, with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,917 genes, with 379 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.

scholarly journals The Genome Sequence of the Tomato-Pathogenic Actinomycete Clavibacter michiganensis subsp. michiganensis NCPPB382 Reveals a Large Island Involved in Pathogenicity

2008 ◽  
Vol 190 (6) ◽  
pp. 2138-2149 ◽  
Author(s):  
Karl-Heinz Gartemann ◽  
Birte Abt ◽  
Thomas Bekel ◽  
Annette Burger ◽  
Jutta Engemann ◽  
...  
Keyword(s):  
Serine Proteases ◽  
Clavibacter Michiganensis ◽  
Reduced Sulfur Compounds ◽  
Apparent Lack ◽  
Clavibacter Michiganensis Subsp ◽  
Related Organism ◽  
Large Island ◽  
Genes Encoding ◽  
Insertion Elements ◽  
Small Stable Rna

ABSTRACT Clavibacter michiganensis subsp. michiganensis is a plant-pathogenic actinomycete that causes bacterial wilt and canker of tomato. The nucleotide sequence of the genome of strain NCPPB382 was determined. The chromosome is circular, consists of 3.298 Mb, and has a high G+C content (72.6%). Annotation revealed 3,080 putative protein-encoding sequences; only 26 pseudogenes were detected. Two rrn operons, 45 tRNAs, and three small stable RNA genes were found. The two circular plasmids, pCM1 (27.4 kbp) and pCM2 (70.0 kbp), which carry pathogenicity genes and thus are essential for virulence, have lower G+C contents (66.5 and 67.6%, respectively). In contrast to the genome of the closely related organism Clavibacter michiganensis subsp. sepedonicus, the genome of C. michiganensis subsp. michiganensis lacks complete insertion elements and transposons. The 129-kb chp/tomA region with a low G+C content near the chromosomal origin of replication was shown to be necessary for pathogenicity. This region contains numerous genes encoding proteins involved in uptake and metabolism of sugars and several serine proteases. There is evidence that single genes located in this region, especially genes encoding serine proteases, are required for efficient colonization of the host. Although C. michiganensis subsp. michiganensis grows mainly in the xylem of tomato plants, no evidence for pronounced genome reduction was found. C. michiganensis subsp. michiganensis seems to have as many transporters and regulators as typical soil-inhabiting bacteria. However, the apparent lack of a sulfate reduction pathway, which makes C. michiganensis subsp. michiganensis dependent on reduced sulfur compounds for growth, is probably the reason for the poor survival of C. michiganensis subsp. michiganensis in soil.

TAS49—a dispersed repetitive sequence isolated from subtelomeric regions of Nicotiana tomentosiformis chromosomes

Genome ◽  
2000 ◽  
Vol 43 (2) ◽  
pp. 273-284
Author(s):  
Mirka Horáková ◽  
Jirí Fajkus
Keyword(s):  
Dna Sequences ◽  
Repetitive Sequence ◽  
Repetitive Sequences ◽  
Reading Frame ◽  
Diploid Genome ◽  
Nicotiana Tomentosiformis ◽  
Dna Repeat ◽  
The Family ◽  
A Subunit ◽  
Subtelomeric Regions

We have isolated and characterized a new repetitive sequence, TAS49, from terminal restriction fragments of Nicotiana tomentosiformis genomic DNA by means of a modified vectorette approach. The TAS49 was found directly attached to telomeres of N. tabacum and one of its ancestors, N. tomentosiformis, and also at inner chromosome locations. No association with telomeres was detected neither in N. otophora nor in the second tobacco ancestor, N. sylvestris. PCR and Southern hybridization reveal similarities in the arrangement of TAS49 on the chromosomes of 9 species of the genus Nicotiana, implying its occurrence as a subunit of a conserved complex DNA repeat. TAS49 belongs to the family of dispersed repetitive sequences without features of transposons. The copy number of TAS49 varies widely in the genomes of 8 species analyzed being lowest in N. sylvestris, with 3300 copies per diploid genome. In N. tomentosiformis, TAS49 forms about 0.56% of the diploid genome, corresponding to 17 400 copies. TAS49 units are about 460 bp long and show about 90% of mutual homology, but no significant homology to DNA sequences deposited in GenBank and EMBL. Although genomic clones of TAS49 contain an open reading frame encoding a proline-rich protein similar to plant extensins, no mRNA transcript was detected. TAS49 is extensively methylated at CpG and CpNpG sites and its chromatin forms nucleosomes phased with a 170 ± 8 bp periodicity. Key words: repetitive DNA sequence, subtelomere, plant, Nicotiana.

scholarly journals Expression of tissue-specific Ren-1 and Ren-2 genes of mice: comparative analysis of 5'-proximal flanking regions.

1984 ◽  
Vol 4 (11) ◽  
pp. 2321-2331 ◽  
Author(s):  
L J Field ◽  
W M Philbrick ◽  
P N Howles ◽  
D P Dickinson ◽  
R A McGowan ◽  
...  
Keyword(s):  
Structural Gene ◽  
Transcription Initiation ◽  
Submaxillary Gland ◽  
Inbred Strains ◽  
Initiation Site ◽  
Sequence Motifs ◽  
Specific Expression ◽  
Tissue Specific ◽  
Reading Frame ◽  
A Minor

All inbred strains of mice carry the Ren-1 structural gene, which encodes the renin-1 isozyme, the classical renin activity found in kidneys. In addition, some strains carry a second renin structural gene, Ren-2, which encodes the predominantly expressed submaxillary gland renin isozyme, renin-2. Ren-1 and Ren-2 exhibit markedly different patterns of tissue-specific expression. In an effort to understand the molecular basis for this differential expression, detailed analysis of the genomic sequences corresponding to the Ren-1 and Ren-2 genes, and the transcripts originating from these loci, was undertaken. Sequence analysis of regions proximal to the structural genes indicated the presence of eucaryotic consensus sequences for transcription. These sequence motifs were strongly conserved between Ren-1 and Ren-2. Approximately 150 bases upstream from the major transcription initiation site, significant differences between these genes were apparent, including the presence of a repetitive DNA element in the Ren-2 copy as well as other breaks in homology and sequence curiosities. Strong homology between Ren-1 and Ren-2 resumed at a point ca. 200 bases further upstream on Ren-1. S1 analysis of submaxillary gland and kidney RNA populations indicated that the majority of transcripts initiate at homologous positions on Ren-1 and Ren-2. On a per cell basis, the accumulation of Ren-1 transcripts in the kidney and Ren-2 transcripts in the submaxillary gland are probably equivalent. These results suggest that it is tissue-specific utilization of the homologous start sites that is critical to their differential patterns of expression. Models which can account for this observation are presented. Interestingly, we found a minor fraction of transcripts initiating 5' to the major transcription start site. These transcripts encoded an open reading frame which may add an additional 23 amino acids to the N-terminus of the renin precursor.

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