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.

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.

scholarly journals Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii

1989 ◽  
Vol 86 (17) ◽  
pp. 6449-6453 ◽  
Author(s):  
E Fernández ◽  
R Schnell ◽  
L P Ranum ◽  
S C Hussey ◽  
C D Silflow ◽  
...  
Keyword(s):  
Nitrate Reductase ◽  
Chlamydomonas Reinhardtii ◽  
Structural Gene ◽  
Genomic Library ◽  
Regulatory Gene ◽  
Amino Acid Level ◽  
Cdna Probe ◽  
Isolation And Characterization ◽  
Cloned Gene ◽  
In Cells

The nitrate reductase structural gene of Chlamydomonas reinhardtii has been isolated from a genomic library by using a nitrate reductase cDNA probe from barley. Restriction fragment length polymorphism analyses mapped the Chlamydomonas clone (B6a) to the nitrate reductase structural gene locus nit-1. Overlapping inserts cover a region of the genome of about 24 kilobases containing the entire gene, which spans approximately 5-8 kilobases. Sequence analysis of DNA fragments from the B6a clone demonstrated a high degree of sequence similarity at the amino acid level with regions corresponding to portions of the heme and FAD/NADH-binding domains of tobacco and Arabidopsis thaliana nitrate reductases and human NADH cytochrome b5 reductase. The identity of the cloned gene as nitrate reductase was confirmed by its ability to complement a nit-1 mutation upon transformation. The nitrate reductase gene produced a 3.4-kilobase transcript in cells derepressed with nitrate; the transcript was undetectable in cells grown in the presence of ammonium. In cells that contain a mutation in the putative regulatory gene nit-2, significantly lower levels of the 3.4-kilobase transcript were found, indicating that the wild-type nit-2 gene is involved in the control of nitrate reductase transcript levels.

Isolation and characterization of a developmentally regulated homeobox sequence in the Mexican axolotl Ambystoma mexicanum

1990 ◽  
Vol 68 (3) ◽  
pp. 622-629 ◽  
Author(s):  
Mary Whiteley ◽  
John B. Armstrong
Keyword(s):  
Genomic Dna ◽  
Genomic Library ◽  
Ambystoma Mexicanum ◽  
Tail Bud ◽  
Developmentally Regulated ◽  
Mexican Axolotl ◽  
Isolation And Characterization ◽  
Partial Genomic Library ◽  
Homeobox Sequence

A homeobox-containing genomic DNA fragment was isolated from the Mexican axolotl. This clone was obtained from a partial genomic library enriched for sequences that cross-hybridized with the Drosophila Antp homeobox under low stringency hybridization conditions. DNA sequence analysis revealed that this sequence (Ahox1) was 66% homologous to the Antp homeobox sequence and was most closely related to the mouse Hox-1.6 (84% identity) and Drosophila lab (79% identity) homeobox sequences. Several cross-hybridizing fragments to Ahox1 were detected in both mouse and axolotl genomic DNA. This sequence was also shown to be conserved in other Ambystoma species. Northern blot analysis revealed that genes containing this sequence are developmentally regulated. Transcripts hybridizing to the Ahox1 homeobox probe were detected during the neurula and tail bud stages of development.Key words: axolotl, homeobox, mouse, Drosophila, gene expression.

Isolation and characterization of SSR sequences from the genome and TAC clones of common wheat using the PCR technique

Genome ◽  
2006 ◽  
Vol 49 (5) ◽  
pp. 432-444 ◽  
Author(s):  
Michiya Koike ◽  
Kanako Kawaura ◽  
Yasunari Ogihara ◽  
Atsushi Torada
Keyword(s):  
Ssr Markers ◽  
Common Wheat ◽  
Genomic Dna ◽  
Genomic Library ◽  
Artificial Chromosome ◽  
Triticum Aestivum L ◽  
Pcr Screening ◽  
Artificial Chromosomes ◽  
Isolation And Characterization ◽  
Simple Sequence

We have developed the 2-step PCR method, a kind of suppression PCR procedure, to isolate simple sequence repeats (SSRs) from common wheat (Triticum aestivum L.) in a more convenient manner. This system requires neither genomic library screening nor the SSR-enrichment procedure. As a result, we designed 131 primer pairs based on isolated SSRs from not only genomic DNA, but also transformation-competent artificial chromosome (TAC) clones. It has been demonstrated that 34 of the 131 SSR markers developed were polymorphic among 8 wheat lines. Four of 34 polymorphic SSR markers were derived from TAC clones, indicating that this method could be applied to the targeted development of unique SSR markers in large genomic DNA libraries such as those composed of bacterial artificial chromosomes (BACs). A considerable number of isolated SSR clones had similarities with part of several long terminal repeats of retrotransposons (LTR-RTs) identified in various Triticeae genome sequences. Most of those SSRs showed smear amplification profiles, suggesting that a considerable number of dysfunctional SSRs originating from repetitive DNA components, especially LTR-RTs, might exist in the common wheat genome.Key words: common wheat, simple sequence repeat (SSR), PCR screening, LTR-retrotransposon, TAC clone.

Protease B of Saccharomyces cerevisiae: Isolation and Regulation of the PRB1 Structural Gene

Genetics ◽  
1987 ◽  
Vol 115 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Charles M Moehle ◽  
Martha W Aynardi ◽  
Michael R Kolodny ◽  
Frances J Park ◽  
Elizabeth W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Structural Gene ◽  
Genomic Library ◽  
Time Lag ◽  
Proteolytic Processing ◽  
Coding Region ◽  
Protease B ◽  
Endonuclease Mapping ◽  
Protein Molecular Weight

ABSTRACT We have isolated the structural gene, PRB1, for the vacuolar protease B of Saccharomyces cerevisiae from a genomic library by complementation of the prb1-1122 mutation. Deletion analysis localized the complementing activity to a 3.2-kilobase pair XhoI-HindIII restriction enzyme fragment. The fragment was used to identify a 2.3-kilobase mRNA. S1 endonuclease mapping indicated that the mRNA and the gene were colinear. No introns were detected. The mRNA is of sufficient size to encode a protein of about 69,000 molecular weight, a number much larger than either the mature enzyme (≃30,000 protein molecular weight) or the sole reported precursor (≃39,000 protein molecular weight). These results suggest that proteolytic processing steps beyond that thought to be catalyzed by protease A may be required to convert the initial glycosylated translation product into mature protease B. The PRB1 mRNA is made in substantial amounts only when the cells have exhausted the glucose supply and enter the diauxic plateau. There is an extended time lag between PRB1 transcription and expression of protease B activity. A deletion that removes about 83% of the coding region was constructed as a diploid heterozygote. Spores bearing the deletion germinate, grow at normal rates into colonies, and have no obvious phenotype beyond protease B deficiency.

Overexpression of the dctA gene in Rhizobium meliloti: effect on transport of C4 dicarboxylates and symbiotic nitrogen fixation

1992 ◽  
Vol 38 (6) ◽  
pp. 555-562 ◽  
Author(s):  
Vipin Rastogi ◽  
Monika Labes ◽  
Turlough Finan ◽  
Robert Watson
Keyword(s):  
Nitrogen Fixation ◽  
Acetylene Reduction ◽  
Symbiotic Nitrogen Fixation ◽  
Plasmid Stability ◽  
Rhizobium Meliloti ◽  
Mrna Synthesis ◽  
Dicarboxylate Transport ◽  
Fusion Strain ◽  
Tryptophan Operon ◽  
Specific Mrna

Symbiotic nitrogen fixation may be limited by the transport of C4 dicarboxylates into bacteroids in the nodule for use as a carbon and energy source. In an attempt to increase dicarboxylate transport, a plasmid was constructed in which the Rhizobium meliloti structural transport gene dctA was fused to a tryptophan operon promoter from Salmonella typhimurium, trpPO. This resulted in a functional dctA gene that was no longer under the control of the dctBD regulatory genes, but the recombinant plasmid was found to be unstable in R. meliloti. To stably integrate the trpPO-dctA fusion, it was recloned into pBR325 and recombined into the R. meliloti exo megaplasmid in the dctABD region. The resultant strain showed constitutive dctA-specific mRNA synthesis which was about 5-fold higher than that found in fully induced wild-type cells. Uptake assays showed that [14C]succinate transport by the trpPO-dctA fusion strain was constitutive, and the transport rate was the same as that of induced control cells. Acetylene reduction assays indicated a significantly higher rate of nitrogen fixation in plants inoculated with the trpPO-dctA fusion strain compared with the control. Despite this apparent increase, the plants had the same top dry weights as those inoculated with control cells. Key words: acetylene reduction, genetic engineering, nodule, plasmid stability, promoter.

Molecular cloning of the murine adenosine deaminase gene from a genetically enriched source: identification and characterization of the promoter region

1986 ◽  
Vol 6 (12) ◽  
pp. 4458-4466
Author(s):  
D E Ingolia ◽  
M R Al-Ubaidi ◽  
C Y Yeung ◽  
H A Bigo ◽  
D Wright ◽  
...  
Keyword(s):  
Adenosine Deaminase ◽  
Structural Gene ◽  
Transcription Initiation ◽  
Genomic Library ◽  
Gene Promoter ◽  
Mouse Cell ◽  
Gene Promoters ◽  
Sp1 Transcription Factor ◽  
Base Pair Fragment ◽  
Functional Analyses

A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplified copies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clones encompassing the entire ADA structural gene were isolated from this genomic library and used for subsequent structural and functional analyses. Nuclease protection and primer extension analyses served to identify the location of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity was found by functional analyses to reside within a 240-base-pair fragment which contains the transcription initiation sites. Sequences upstream of the transcription initiation sites are very G + C rich (77%) and include a 22 nucleotide stretch of deoxyguanylate residues and two potential Sp1 transcription factor-binding sites. Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that are highly conserved with regard to both sequence content and location and may represent genetic elements which are involved in ADA gene expression.

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