A species-specific oligonucleotide DNA probe for the identification of Meloidogyne incognita

Parasitology ◽  
1991 ◽  
Vol 103 (2) ◽  
pp. 315-319 ◽  
Author(s):  
M. R. Chacon ◽  
R. M. E. Parkhouse ◽  
M. P. Robinson ◽  
P. R. Burrows ◽  
T. Garate
Keyword(s):  
Dna Sequences ◽  
Diagnostic Tool ◽  
Meloidogyne Incognita ◽  
Genomic Dna ◽  
Genomic Library ◽  
Dna Probe ◽  
Equal Length ◽  
Race 1 ◽  
Species Specific

A genomic library of Meloidogyne incognita Race 1 has been prepared in the bacteriophage λgt10 and screened for specific DNA sequences by hybridization with radio-isotope labelled total genomic DNA from a number of Meloidogyne species. One clone isolated (MR1#15), although not totally species specific, clearly showed preferential hybridization to M. incognita. Following subcloning and sequencing of the 255 bp insert, four stretches of the sequence corresponding to oligonucleotides of approximately equal length (~60 bp) were synthesized and examined for specificity. One of them, MR1#15.2, showed the necessary specificity to be used as a diagnostic tool.

Isolation of a yeast gene involved in species-specific pre-tRNA processing

1988 ◽  
Vol 8 (12) ◽  
pp. 5140-5149
Author(s):  
S S Wang ◽  
A K Hopper
Keyword(s):  
Dna Sequences ◽  
Genomic Library ◽  
Nonsense Suppression ◽  
Suppressor Activity ◽  
Specific Product ◽  
Trna Processing ◽  
Trna Splicing ◽  
Trna Species ◽  
Multiple Copies ◽  
Species Specific

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.

Molecular and cytogenetic analysis of repetitive DNA in pea (Pisum sativum L.)

Genome ◽  
2001 ◽  
Vol 44 (4) ◽  
pp. 716-728 ◽  
Author(s):  
Pavel Neumann ◽  
Marcela Nouzová ◽  
Jirí Macas
Keyword(s):  
Pisum Sativum ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Tandem Repeats ◽  
Genomic Library ◽  
Chromosome Morphology ◽  
Plant Genome ◽  
Genomic Repeats ◽  
Dispersed Repeats

A set of pea DNA sequences representing the most abundant genomic repeats was obtained by combining several approaches. Dispersed repeats were isolated by screening a short-insert genomic library using genomic DNA as a probe. Thirty-two clones ranging from 149 to 2961 bp in size and from 1000 to 39 000/1C in their copy number were sequenced and further characterized. Fourteen clones were identified as retrotransposon-like sequences, based on their homologies to known elements. Fluorescence in situ hybridization using clones of reverse transcriptase and integrase coding sequences as probes revealed that corresponding retroelements were scattered along all pea chromosomes. Two novel families of tandem repeats, named PisTR-A and PisTR-B, were isolated by screening a genomic DNA library with Cot-1 DNA and by employing genomic self-priming PCR, respectively. PisTR-A repeats are 211–212 bp long, their abundance is 2 × 104 copies/1C, and they are partially clustered in a secondary constriction of one chromosome pair with the rest of their copies dispersed on all chromosomes. PisTR-B sequences are of similar abundance (104 copies/1C) but differ from the "A" family in their monomer length (50 bp), high A/T content, and chromosomal localization in a limited number of discrete bands. These bands are located mainly in (sub)telomeric and pericentromeric regions, and their patterns, together with chromosome morphology, allow discrimination of all chromosome types within the pea karyotype. Whereas both tandem repeat families are mostly specific to the genus Pisum, many of the dispersed repeats were detected in other legume species, mainly those in the genus Vicia.Key words: repetitive DNA, plant genome, retroelements, satellite DNA, Pisum sativum.

Isolation and characterization of six different chicken actin genes

1984 ◽  
Vol 4 (11) ◽  
pp. 2498-2508
Author(s):  
K S Chang ◽  
W E Zimmer ◽  
D J Bergsma ◽  
J B Dodgson ◽  
R J Schwartz
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Smooth Muscle Actin ◽  
Actin Gene ◽  
Muscle Actin ◽  
Repeated Dna ◽  
Alpha Smooth Muscle Actin ◽  
Actin Genes ◽  
Actin Isoform

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

scholarly journals Isolation of a yeast gene involved in species-specific pre-tRNA processing.

1988 ◽  
Vol 8 (12) ◽  
pp. 5140-5149 ◽  
Author(s):  
S S Wang ◽  
A K Hopper
Keyword(s):  
Dna Sequences ◽  
Genomic Library ◽  
Nonsense Suppression ◽  
Suppressor Activity ◽  
Specific Product ◽  
Trna Processing ◽  
Trna Splicing ◽  
Trna Species ◽  
Multiple Copies ◽  
Species Specific

To identify genes involved in pre-tRNA processing, we searched for yeast DNA sequences that specifically enhanced the expression of the SUP4(G37) gene. The SUP4(G37) gene possesses a point mutation at position 37 of suppressor tRNA(Tyr). This lesion results in a reduced rate of pre-tRNA splicing and a decreased level of nonsense suppression. A SUP4(G37) strain was transformed with a yeast genomic library, and the transformants were screened for increased suppressor activity. One transformant contained a plasmid that encoded an unessential gene, STP1, that in multiple copies enhanced the suppression of SUP4(G37) and caused increased production of mature SUP4(G37) product. Disruption of the genomic copy of STP1 resulted in a reduced efficiency of SUP4-mediated suppression and the accumulation of pre-tRNAs. Not all intron-containing pre-tRNAs were affected by the stp1-disruption. At least five of the nine families of pre-tRNAs were affected. Two other species, pre-tRNA(Ile) and pre-tRNA(3Leu), were not. We propose that STP1 encodes a tRNA species-specific product that functions as a helper for pre-tRNA splicing. The STP1 product may interact with pre-tRNAs to generate a structure that is efficiently recognized by splicing machinery.

scholarly journals Isolation and characterization of six different chicken actin genes.

1984 ◽  
Vol 4 (11) ◽  
pp. 2498-2508 ◽  
Author(s):  
K S Chang ◽  
W E Zimmer ◽  
D J Bergsma ◽  
J B Dodgson ◽  
R J Schwartz
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Smooth Muscle Actin ◽  
Actin Gene ◽  
Muscle Actin ◽  
Repeated Dna ◽  
Alpha Smooth Muscle Actin ◽  
Actin Genes ◽  
Actin Isoform

Genes representing six different actin isoforms were isolated from a chicken genomic library. Cloned actin cDNAs as well as tissue-specific mRNAs enriched in different actin species were used as hybridization probes to group individual actin genomic clones by their relative thermal stability. Restriction maps showed that these actin genes were derived from separate and nonoverlapping regions of genomic DNA. Of the six isolated genes, five included sequences from both the 5' and 3' ends of the actin-coding area. Amino acid sequence analysis from both the NH2- and COOH-terminal regions provided for the unequivocal identification of these genes. The striated isoforms were represented by the isolated alpha-skeletal, alpha-cardiac, and alpha-smooth muscle actin genes. The nonmuscle isoforms included the beta-cytoplasmic actin gene and an actin gene fragment which lacked the 5' coding and flanking sequence; presumably, this region of DNA was removed from this gene during construction of the genomic library. Unexpectedly, a third nonmuscle chicken actin gene was found which resembled the amphibian type 5 actin isoform (J. Vandekerckhove, W. W. Franke, and K. Weber, J. Mol. Biol., 152:413-426). This nonmuscle actin type has not been previously detected in warm-blooded vertebrates. We showed that interspersed, repeated DNA sequences closely flanked the alpha-skeletal, alpha-cardiac, beta-, and type 5-like actin genes. The repeated DNA sequences which surround the alpha-skeletal actin-coding regions were not related to repetitious DNA located on the other actin genes. Analysis of genomic DNA blots showed that the chicken actin multigene family was represented by 8 to 10 separate coding loci. The six isolated actin genes corresponded to 7 of 11 genomic EcoRI fragments. Only the alpha-smooth muscle actin gene was shown to be split by an EcoRI site. Thus, in the chicken genome each actin isoform appeared to be encoded by a single gene.

Differential Diagnosis of Taenia saginata and Taenia solium Infection by PCR

2000 ◽  
Vol 38 (2) ◽  
pp. 737-744 ◽  
Author(s):  
Luis Miguel González ◽  
Estrella Montero ◽  
Leslie J. S. Harrison ◽  
R. Michael E. Parkhouse ◽  
Teresa Garate
Keyword(s):  
Repetitive Sequence ◽  
Genomic Dna ◽  
Genomic Library ◽  
Taenia Solium ◽  
Differential Detection ◽  
Taenia Saginata ◽  
Noncoding Dna ◽  
Dna Fragment ◽  
Specific Fragment ◽  
Species Specific

We have designed species-specific oligonucleotides which permit the differential detection of two species of cestodes, Taenia saginata and Taenia solium. The oligonucleotides contain sequences established for two previously reported, noncoding DNA fragments cloned from a genomic library of T. saginata. The first, which is T. saginata specific (fragment HDP1), is a repetitive sequence with a 53-bp monomeric unit repeated 24 times in direct tandem along the 1,272-bp fragment. From this sequence the two oligonucleotides that were selected (oligonucleotides PTs4F1 and PTs4R1) specifically amplified genomic DNA (gDNA) from T. saginata but not T. solium or other related cestodes and had a sensitivity down to 10 pg of T. saginata gDNA. The second DNA fragment (fragment HDP2; 3,954 bp) hybridized to bothT. saginata and T. solium DNAs and was not a repetitive sequence. Three oligonucleotides (oligonucleotides PTs7S35F1, PTs7S35F2, and PTs7S35R1) designed from the sequence of HDP2 allowed the differential amplification of gDNAs from T. saginata, T. solium, and Echinococcus granulosus in a multiplex PCR, which exhibits a sensitivity of 10 pg.

scholarly journals Identification of Campylobacter jejuni ATCC 43431-Specific Genes by Whole Microbial Genome Comparisons

2004 ◽  
Vol 186 (14) ◽  
pp. 4781-4795 ◽  
Author(s):  
Frédéric Poly ◽  
Deborah Threadgill ◽  
Alain Stintzi
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Open Reading Frames ◽  
Dna Fragments ◽  
Helicobacter Hepaticus ◽  
Type Iv ◽  
Novel Approach ◽  
The Mean ◽  
Reading Frames

ABSTRACT This study describes a novel approach to identify unique genomic DNA sequences from the unsequenced strain C. jejuni ATCC 43431 by comparison with the sequenced strain C. jejuni NCTC 11168. A shotgun DNA microarray was constructed by arraying 9,600 individual DNA fragments from a C. jejuni ATCC 43431 genomic library onto a glass slide. DNA fragments unique to C. jejuni ATCC 43431 were identified by competitive hybridization to the array with genomic DNA of C. jejuni NCTC 11168. The plasmids containing unique DNA fragments were sequenced, allowing the identification of up to 130 complete and incomplete genes. Potential biological roles were assigned to 66% of the unique open reading frames. The mean G+C content of these unique genes (26%) differs significantly from the G+C content of the entire C. jejuni genome (30.6%). This suggests that they may have been acquired through horizontal gene transfer from an organism with a G+C content lower than that of C. jejuni. Because the two C. jejuni strains differ by Penner serotype, a large proportion of the unique ATCC 43431 genes encode proteins involved in lipooligosaccharide and capsular biosynthesis, as expected. Several unique open reading frames encode enzymes which may contribute to genetic variability, i.e., restriction-modification systems and integrases. Interestingly, many of the unique C. jejuni ATCC 43431 genes show identity with a possible pathogenicity island from Helicobacter hepaticus and components of a potential type IV secretion system. In conclusion, this study provides a valuable resource to further investigate Campylobacter diversity and pathogenesis.

Molecular characterization and chromosomal distribution of species-specific repetitive DNA sequences from Beta corolliflora, a wild relative of sugar beet

Genome ◽  
2000 ◽  
Vol 43 (6) ◽  
pp. 1073-1080 ◽  
Author(s):  
D Gao ◽  
T Schmidt ◽  
C Jung
Keyword(s):  
Sugar Beet ◽  
Repetitive Dna ◽  
Dna Sequences ◽  
Genomic Dna ◽  
Southern Hybridization ◽  
Repetitive Dna Sequences ◽  
Wild Relative ◽  
Chromosomal Distribution ◽  
Species Specific ◽  
Specific Sequences

Repetitive DNA sequences have been isolated from a Sau3AI plasmid library of tetraploid Beta corolliflora (2n = 4x = 36), a wild relative of sugar beet (B. vulgaris). The library was screened by differential hybridization with genomic DNA of B. corolliflora and B. vulgaris. When used as probes for Southern hybridization of genomic DNA, six clones were determined to represent highly repetitive DNA families present only in the B. corolliflora genome. Five other sequences were highly repetitive in B. corolliflora and low or single copy in B. vulgaris. The insert size varied between 43 bp and 448 bp. Two sequences pBC1279 and pBC1944 displayed strong homology to a previously cloned satellite DNA from B. nana. With one exception, sequences are tandemly arranged as revealed by a typical ladder pattern after genomic Southern hybridization. The chromosomal distribution of five probes was determined by fluorescence in situ hybridization (FISH) of mitotic metaphases from B. corolliflora and a triploid hybrid between B. vulgaris and B. corolliflora. Three sequences were spread along all chromosome arms of B. corolliflora while one sequence was present on only six chromosomes. The chromosome-specific sequence pBC216 was found in close vicinity to the 5S rDNA located on B. corolliflora chromosome IV. This set of species-specific sequences has the potential to be used as probes for the identification of monosomic alien addition lines and for marker-assisted gene transfer from wild beet to cultivated beet.Key words: Beta vulgaris, FISH, repetitive DNA, species-specific sequences.

Isolation and characterization ofAzospirillum lipoferumlocus that complementsRhizobium meliloti dctAanddctBmutations

1996 ◽  
Vol 42 (5) ◽  
pp. 503-506 ◽  
Author(s):  
A. K. Tripathi ◽  
B. M. Mishra
Keyword(s):  
Structural Gene ◽  
Genomic Dna ◽  
Genomic Library ◽  
Rhizobium Meliloti ◽  
Dna Probe ◽  
Azospirillum Lipoferum ◽  
Gene Complementation ◽  
Dicarboxylate Transport ◽  
Isolation And Characterization

A DNA probe containing the structural gene for dicarboxylate transport (dctA) of Rhizobium meliloti hybridized strongly with the fragments of Azospirillum lipoferum genomic DNA. A genomic library of A. lipoferum was screened for the dctA gene by complementation of a dctA mutant of Rhizobium meliloti. A recombinant cosmid, p37D, capable of restoring growth of the dctA mutant on dicarboxylates was isolated and found to hybridize to the dctA probe. The ability of p37D to complement the dctB mutant of R. meloliti indicated that dctA and dctB genes in A. lipoferum may be organized adjacent to each other.Key words: Azospirillum lipoferum, dicarboxylate transport gene, complementation cloning.

scholarly journals Novel ‘Bacteriospray’ Method Facilitates the Functional Screening of Metagenomic Libraries for Antimicrobial Activity

2019 ◽  
Vol 2 (1) ◽  
pp. 4 ◽  
Author(s):  
Anissa Brahami ◽  
Annie Castonguay ◽  
Éric Déziel
Keyword(s):  
Dna Sequences ◽  
Genomic Dna ◽  
Genomic Library ◽  
Agar Plate ◽  
Environmental Dna ◽  
Functional Screening ◽  
Genomic Libraries ◽  
Metagenomic Libraries ◽  
Interesting Approach ◽  
Genomic Regions

Metagenomic techniques, notably the cloning of environmental DNA (eDNA) into surrogate hosts, have given access to the genome of uncultured bacteria. However, the determination of gene functions based on DNA sequences alone remains a significant challenge. The functional screening of metagenomic libraries represents an interesting approach in the discovery of microbial metabolites. We describe here an optimized screening approach that facilitates the identification of new antimicrobials among large metagenomic libraries. Notably, we report a detailed genomic library construction protocol using Escherichia coli DH10B as a surrogate host, and demonstrate how vector/genomic DNA dephosphorylation, ligase inactivation, dialysis of the ligation product and vector/genomic DNA ratio greatly influence clone recovery. Furthermore, we describe the use of an airbrush device to screen E. coli metagenomic libraries for their antibacterial activity against Staphylococcus aureus, a method we called bacteriospray. This bacterial spraying tool greatly facilitates and improves the functional screening of large genomic libraries, as it conveniently allows the production of a thinner and more uniform layer of target bacteria compared to the commonly used overlay method, resulting in the screening of 5–10 times more clones per agar plate. Using the Burkholderia thailandensis E264 genomic DNA as a proof of concept, four clones out of 70,000 inhibited the growth of S. aureus and were found to each contain a DNA insert. Analysis of these chromosomic fragments revealed genomic regions never previously reported to be responsible for the production of antimicrobials, nor predicted by bioinformatics tools.

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