HIBRIDACION SOUTHERN BLOT PDF

In molecular biology , a hybridization probe is a fragment of DNA or RNA of variable length usually — bases long which can be radioactively or fluorescently labeled. The probe thereby hybridizes to single-stranded nucleic acid DNA or RNA whose base sequence allows probe—target base pairing due to complementarity between the probe and target. To detect hybridization of the probe to its target sequence, the probe is tagged or "labeled" with a molecular marker of either radioactive or more recently fluorescent molecules; commonly used markers are 32 P a radioactive isotope of phosphorus incorporated into the phosphodiester bond in the probe DNA or digoxigenin , which is a non-radioactive, antibody -based marker. DNA sequences or RNA transcripts that have moderate to high sequence similarity to the probe are then detected by visualizing the hybridized probe via autoradiography or other imaging techniques. Normally, either X-ray pictures are taken of the filter, or the filter is placed under UV light. Detection of sequences with moderate or high similarity depends on how stringent the hybridization conditions were applied—high stringency, such as high hybridization temperature and low salt in hybridization buffers, permits only hybridization between nucleic acid sequences that are highly similar, whereas low stringency, such as lower temperature and high salt, allows hybridization when the sequences are less similar.

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E-mail: banco cigb. The gene encoding cellobiose dehydrogenase from Phanerochaete chrysosporium K3 was isolated through PCR and its bp nucleotide sequence determined. Two distinct 5' flanking sequences were identified by Southern blot hybridization, both having a common bp region, but one of them interrupted by a bp transposon-like element located at bp upstream from the initial ATG.

This element, named Pcc2 , carried 14 bp inverted terminal repeats while lacking the conserved transposase motif it is flanked by a 2 bp GT target duplication site and is present at a high copy number throughout the P.

Keywords : cellobiose dehydrogenase CDH , gene, Phanerochaete chrysosporium , transposon-like element. The white rot fungus Phanerochaete chrysosporium is one of the most efficient wood degrading fungi yet identified [1, 2]. In the last ten years this microorganism has been extensively characterized for biotechnological approaches based on a selective degradation of lignin and aromatic pollutants [3, 4].

The cellobiose dehydrogenase CDH enzyme is one interesting element of this degradation system and it is secreted during the induction of the P. CDH is a hemoflavoprotein which oxides cellobiose and higher cellodextrins to their corresponding lactones. Molecular oxygen may function as an electron acceptor producing hydrogen peroxide, but other compounds like quinones, ferricyanide and triodide ions are better [7].

The biological function of CDH is unknown, but several suggestions have been presented [8, 9]. Substantial variation has been observed among P.

Here, during the isolation of the CDH promoter region, we detected an inserted DNA fragment which seems to be a transposon-like element. Transposable elements TEs are ancient and ubiquitous components of fungal genomes [10] and make up a substantial proportion of the total DNA in most, if not all, eukaryotic genomes [11]. Some of these elements have been shown to actively affect gene structure and function in several ways: inactivation of gene expression upon insertion, modification of the nucleotide sequence through excision, and probably by inducing extensive chromosomal rearrangements [10].

There are two main classes of TEs: the retrotransposons, that transpose through a process involving reverse transcription, and the transposons that move by an excision-insertion mechanism [11]. Here we report the isolation of the 5' non-coding region of the CDH gene and the identification of a transposon like-element inserted in this region.

For the isolation of chromosomal DNA, spores of P. The mycelium was harvested, washed with water and the genomic DNA extracted as described by Raeder and Broda [29]. DNA manipulations, including restriction digests, agarose gel electrophoresis, ligations, transformations of E. Plasmid pBS3. Ten nanograms of each plasmid were subjected to 30 cycles of amplification by PCR under the same reaction conditions as described above.

Ten micrograms of genomic DNA from P. The digestion products were separated by agarose gel electrophoresis, transferred to a nylon membrane Biodyne A transfer membrane, Pall Biosupport membranes and hybridized with probe 1 and with probe 2 in order to identify the zone corresponding to the promoter region. Between the two hybridizations the filter was stripped for the removal of the probe.

The filter was washed twice in 2X SCC, 0. DNA fragments of about 1. Approximately and transformant colonies were screened for plasmids containing the 1.

Two plasmids containing the predicted 1. In order to identify the insertion occurring in the genome, the same previously used membrane was stripped and re-hybridized with a 2 Kb fragment from pBS3. Double-stranded sequencing of the pBS1. Primers Pharmacia were used to walk progressively along both DNA strands. The gene sequence was similar to the previously published cDNA sequence of this fungus [25], and it corresponds exactly to the cDNA sequence reported by Li , et al.

The Bam H I site is located at bp in the genomic clone, within the fourth intron, therefore two fragments or a single fragment longer than bp should be expected when the total DNA is digested to completion with Bam H I and hybridized to probe 1 or 2, respectively, provided that only a single copy of the gene exists in this organism.

However, in the Southern blot experiment three fragments of 1. Two fragments of similar sizes were observed, using Eco R I lane 4 , Eco R V lane 5 and Xba I lane 6 restriction enzymes and both probes in the hybridization. Since the gene does not have internal cut sites for these enzymes, it seems probable that these fragments represent two copies of the CDH gene in P. As shown by digestion with Sal I, which cuts the gene three times, four different size fragments 1.

The 1. Genomic DNA was subjected to double digestion with several pairs of restriction enzymes, and results of hybridization of the resulting fragments with both probes are shown in Figure 1B, lanes This analysis clearly established that there were no differences between the two copies in the coding and 3' flanking regions, and it shows that all differences occur within the 5' flanking region.

A difference of about 2. On the basis of these results, a restriction map of the CDH gene and the flanking regions was constructed Figure 1A. In order to isolate the two 5' upstream regions the 1. The sequence analysis shows that there were no differences in bp of the 5' region of the coding region and in the first bp upstream the ATG.

However, an inserted sequence was detected only in the pBS3. Figure 1A:. The triangle represents a 2. Labeled horizontal bars beneath the maps identify the regions that correspond to probes used in Southern blot hybridization. Figure 1.

In lane 1 is the undigested genomic DNA, in lane 14, 1 ng of the genomic gene isolated with primers 1F-4R probe 1 as the positive control. The DNA was electrophoretically separated on an 0. The same molecular weight markers were run in lanes 8 and 9 and their sizes are shown in kb on both sides. Recently, Li , et al. The cDNA and genomic sequence reported by us coincided with the sequence of cdh-2 , as well as the previous cDNA sequence reported by Li , et al. Sequence analysis of the two isolated 5' flanking regions of the CDH gene revealed a 2.

The complete sequence of the insertion was determined Figure 2. An element was identified that contains perfect inverted terminal repeats ITRs of 14 bp. Nucleotide sequence of the insertion element Pcc2 within the 5' upstream region of the plasmid pBS3. The underlined sequences are the 14 bp inverted terminal repeats ITRs.

The element was flanked on both sides by a GT dinucleotide, only one copy of which occurs at the corresponding site in the 5' upstream ATG region of the gene without the insertion, suggesting a duplication of the target site formed during the insertion. Certain features of the insertion sequence, such as the presence of perfect terminal repeats and the duplication of the target site associated with the insertion are common for many prokaryotic and eukaryotic transposons [11, 34].

The insertion therefore is likely to be a transposable element which we named Pcc2 , although this element does not contain a typical dinucleotide TA target duplication found in most of the fungal transposons related to the Caenorhabditis elegans Tc1 element [35]. Transposons have been found in a number of fungal species and classified in two main classes: class I elements which transposes by reverse transcription of an RNA copy of the element and class II which transposes directly through DNA copies.

Also several non-LTR retrotranposons with structural features of long interspersed nuclear elements have been characterized: Tad in N. Others are members of a new family described so far only in fungi reviewed in 10 , this is the case for Fot1 in F. Other less-characterized TEs are known that may represent new families: Hop in F. Pcc2 has several features which allow to classify it as a transposon-like element, however with the data we now have, it is not possible to find a homology with either class 1 or class 2 of the described TEs families.

We isolated a 2 kb fragment probe 3 by PCR using specific primer oligonucleotides which correspond to the sequence of this element. With this probe and Southern hybridization, we found that this insertion appeared in high copy numbers and randomly dispersed within the genome of P. Figure 3. Distribution of the Pcc2 element in the P.

Southern blot is identical to that shown in Figure 1 B, but hybridized here with the Pcc2 probe probe 3. A database examination and comparison of the Pcc2 sequence revealed no extended open reading frame and as in the first transposon-like element described in P. Other transposons contain large open reading frames which show similarity at the amino acid level to the transposase motif conserved in the Tc1 superfamily [10], but many others such as the Fot1 from F.

The Pce1 element was shown to harbor a bp insertion within the lignin peroxidase allele of P. Insertion of a transposable element within or adjacent to a chromosomal gene can directly alter its expression. In most cases the element blocks expression, but sometimes the effect is subtler. Transposable elements can increase the number of copies of sequences within the genome, either because they are, by chance, recognized as intermediates for replicative transposition events or because they are included in duplications produced by recombination between elements, generating sequence variation within genes that may be evolutionarily advantageous [10].

The presence of this element in the promoter region may affect the expression of the CDH gene and might play a role in genetic variation and evolutionary divergence of this gene in the P. Further studies are underway to examine the possible role of Pcc2 on the inheritance and transcription of the CDH gene. We deeply thank Professor Robert Brambl and Dr.

David Eaker for valuable suggestions and critical reviews of the manuscript. Microbial and enzymatic degradation of wood and wood components. Springer-Verlag Berlin Heidelberg, Germany; Boominathan K, Reddy CA.

Fungal degradation of lignin: Biotechnological Applications. Pp In Arora, D. Fungal Biotechnology. Marcel Dekker Inc. NewYork; Enzymatic "combustion": the microbial degradation of lignin. Annu Rev Microbiol ; Hammel KE. Organopollutant degradation by lignolytic fungi. Enzyme Microb Technol ; Ultrastructural aspect of wood degradation by Sporotrichum pulvurulentum. Observations on spruce wood impregnated with glucose.

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