Rounds Of Subtractive Hybridization Research Articles

CDNA representational difference analysis of the deltamelthrin‐resistant and ‐susceptible populations in diamondback moth (Plutella xylostella L.)

Abstract: The improved cDNA representational difference analysis was preliminarily used to analyse the genetic differences between the deltamethrin‐susceptible and ‐resistant strain of the diamondback moth. The driver amplicon is the cDNA from the susceptible strain while the tester amplicon is from the resistant strain. After four rounds of subtractive hybridization, we obtained one different product, the size of which was about 200 bp. Comparisons between our sequencing results and data in GenBank show that there is one sequence which has a relatively high homology to the ubiquitin gene, but there are no reports indicating that its upregulated expression is correlated with insecticide resistance. Hence further studies are warranted.

Corl1, a Novel Neuronal Lineage-specific Transcriptional Corepressor for the Homeodomain Transcription Factor Lbx1

During development, neuronal identity is determined by a combination of numerous transcription factors. However, the mechanisms of synergistic action of these factors in transcriptional regulation and subsequent cell fate specification are largely unknown. In this study, we identified a novel gene, Corl1, encoding a nuclear protein with homology to the Ski oncoprotein. Corl1 was highly selectively expressed in the central nervous system (CNS). In the embryonic CNS, Corl1 was expressed in a certain subset of postmitotic neurons generated posterior to the midbrain-hindbrain border. In the developing spinal cord, Corl1 was selectively expressed in the dorsal horn interneurons where a homeodomain transcription factor, Lbx1, is required for proper specification. Corl1 was localized in a nuclear dot-like structure and interacted with general transcriptional corepressors. In addition, Corl1 showed transcriptional repression activity in the GAL4-fusion system, indicating its involvement in the regulation of transcriptional repression. Furthermore, Corl1 interacted with Lbx1 and cooperatively repressed transcription, suggesting that it acts as a transcriptional corepressor for Lbx1 in regulating cell fate determination in the dorsal spinal cord. Corl1 corepressor activity did not depend on Gro/TLE activity, and Gro/TLE also functioned as a corepressor for Lbx1. Thus, Lbx1 can select two independent partners, Corl1 and Gro/TLE, as corepressors. Identification of a novel transcriptional corepressor with neuronal subtype-restricted expression might provide insights into the mechanisms of cell fate determination in neurons.

Isolation of pathogen-induced Chinese cabbage genes by subtractive hybridization employing selective adaptor ligation

We have developed a subtractive cloning method in which target sequences are effectively enriched by selective adaptor ligation and PCR after hybridization. In this method both tester and driver DNAs are digested with RsaI, ligated with the linker DNA containing a KpnI recognition site, and amplified by PCR. The tester DNA samples are divided into two aliquots, each digested with either RsaI or KpnI. The two DNA samples are then combined and hybridized with an excess of the driver DNA retaining the linker. After hybridization, the DNA mixture is ligated to a new adaptor compatible only with double-stranded tester/tester DNAs. Therefore, only the tester/tester is selectively amplified in subsequent PCR. This also leads to complete elimination of the tester DNA hybridized with driver DNA from the tester DNA population. Although our protocol employs enzymatic treatments, the efficiency of the enzymatic treatments does not affect the subtraction efficiency. This new subtractive enrichment method was applied to isolate Chinese cabbage defense-related genes induced by Pseudomonas syringae pv. tomato ( Pst), which elicits a hypersensitive response in Chinese cabbage. After two or three rounds of subtractive hybridization, the sequences of enriched DNAs were determined and examined by BLAST analysis. Northern blot hybridization showed that 12 of the 19 genes analyzed were strongly induced by Pst treatment. Among the 12 Pst-induced genes five represent pathogenesis-related genes encoding PR1a, two chitinases, a thaumatin-like protein, and a PR4 protein. Other Pst-induced genes include two cytochrome P450 genes responsible for glucosinolate biosynthesis, a disease resistance gene homolog, and several genes encoding proteins with unknown functions.

Direct cloning of full-length cell differentially expressed genes by multiple rounds of subtractive hybridization based on long-distance PCR and magnetic beads.

Subtractive cloning is an ideal technique for identifying genes differentially expressed in two nuclear acids population (). The polymerase chain reaction (PCR)-based subtraction is the method of choice when the starting samples are heterogeneous or difficult to obtain, which often occurs in the tissues to be compared. PCR amplification is the easiest method for generating adequate amount of nuclear acids for multiple-round hybridization. However, the bias in the relative representation of mRNA molecules in the starting materials and the accumulation of shorter fragments become the major deficiencies for this method and should be overcome. The bias caused by PCR amplification is because of the tendency of preferentially amplifying short fragments and certain templates with unique sequences in the sample. The thermophilic polymerase that is optimized to amplify multiple genes would be helpful and the adoption of gel filtration in preparation of templates for amplification could hinder the tendency of short fragment accumulation. In addition, increasing the amount of starting samples would represent much more molecules in tissues.

ISOLATION OF PHASE‐SPECIFIC COMPLE‐MENTARY DNAS FROM CARPOSPOROPHYTES OF GRACILARIOPSIS LEMA‐NEIFORMIS USING MAGNETIC BEADS AND PCR

Kamiya, M.1, Moon, D. A.2, Kawai, H.1 & Goff, L. J.2 1Kobe University Research Center for Inland Seas, 2746 Iwaya, Awaji‐cho 656‐2401 Japan; 2Department of Biology, University of California, Santa Cruz, CA 95064 USAAlthough the morphology and developmental patterns of carposporophyte stage have been investigated well, there are few molecular, genetic, or biochemical data about this stage. The greatest obstacle to this research has been that the conventional methods to isolate tissue‐specific genes require a lot of tissues, but the carposporophyte is very tiny and mostly embedded in female gametophyte tissues. Recent advanced techniques have allowed the subtractive cloning of differentially expressed genes from small amounts of tissue or cells. We applied the subtractive hybridization method using magnetic beads and PCR to the analysis of phase‐specific cDNAs from carpo‐sporophytes of Gracilariopsis lemaneiformis (Gracilariales, Rhodophyta). A hundred cystocarps were dissected to isolate gonimoblast tissues, and total RNAs were extracted from the gonimoblast tissues and the female gametophyte branches, respectively. Messenger RNAs were captured on paramagnetic oligo‐dT beads, followed by first‐strand cDNA synthesis on the beads. Three rounds of subtractive hybridization between the amplified second‐strand carposporophyte cDNA in solution and the first‐strand gametophyte cDNA attached to magnetic beads were sufficient to remove common genes present in both gametophyte and carposporophyte stages. A specific PCR product from the nuclear GAPDH gene was readily amplified from gametophyte and carposporophyte cDNA, but no amplification was observed using the subtracted carposporophyte cDNA as template. This control PCR product demonstrates that the hybridization steps successfully removed the common GAPDH cDNA, which is found in both stages, giving confidence that the remaining genes cloned from the subtracted carposporophyte cDNA library are stage‐specific.

Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA).

Genomic Representational Difference Analysis (gRDA) is a subtractive DNA method to clone the differences between two related genomes, called tester and driver. We have evaluated this method to obtain polymorphic DNA markers for pedigree dogs. Amplified size-selected genomic restriction fragments (amplicons) of two dog littermates were repeatedly hybridized to each other in order to remove (subtract) those restriction fragments common to both sibs. Already after two rounds of subtractive hybridization, a clear enrichment of presumably tester-specific restriction fragments was observed, which was even more pronounced after the third round of subtraction. A plasmid library of 3000 recombinant clones was constructed of the second round and of the third round difference product. DNA sequence determination of randomly chosen clones of each difference product showed that approximately 1000 unique clones were obtained in the second-round difference product and approximately 500 in the third-round difference product. About half of the clones identified in the second-round difference product were also present in the third-round difference product. Of the second-round difference product, 39 different gRDA fragments could be identified, of which 21 were tester specific. In the third-round difference product, 22 different gRDA fragments were identified, of which 18 were tester specific. There were 13 fragments in common, resulting in a total of 48 different fragments. In order to establish the localization of these markers, we performed mapping using the dog radiation hybrid panel RHDF5000. Of 39 mapped clones, 29 were mapped to 20 existing RH groups, and 10 remained unlinked. It is concluded that gRDA is suitable to generate DNA markers to track disease genes within lines of pedigree dogs.

Representational differential analysis detects amplification of satellite sequences in postweaning multisystemic wasting syndrome of pigs.

Representational difference analysis (RDA) was used as a molecular approach to identify unique sequences associated with postweaning multisystemic wasting syndrome (PMWS) in pigs. Three rounds of subtractive hybridization and amplification between driver DNA extracted from normal pigs and tester DNA from PMWS-affected animals were performed. The final product corresponding to sequences associated with PMWS in pigs was analyzed using agarose gel electrophoresis, and 9 fragments were visualized after staining with ethidium bromide. Eight recombinants were successively cloned and sequenced, and the results were then compared with existing databases. Most of the PMWS clones isolated were satellite sequences from pig centrometric regions and 1 was a microsatellite sequence. One clone represented a microsatellite sequence, and 2 clones showed no homology with any gene found in the databases. The sequence comparison data did not reveal any homology with an infectious agent such as a virus or a bacterium. In the present experimental setting, it was concluded that PMWS in pigs triggers molecular changes such as an amplification of genomic regions containing repeated sequences.

20] Suppression subtractive hybridization: A versatile method for identifying differentially expressed genes

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a technique called suppression PCR, and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. As a result only one round of subtractive hybridization is needed and the subtracted library is normalized in terms of abundance of different cDNAs. It dramatically increases the probability of obtaining low-abundance differentially expressed cDNA and simplifies analysis of the subtracted library. The SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes. This chapter provides detailed protocols for the generation of subtracted cDNA and differential screening of subtracted cDNA libraries. As a representative example we demonstrate the usefulness of the method by constructing a testis-specific cDNA library as well as using the subtracted cDNA mixture as a hybridization probe. Finally, we discuss the characteristics of subtracted libraries, the nature and level of background nondifferentially expressed clones in the libraries, as well as a procedure for the rapid identification of truly differentially expressed cDNA clones.

Absence of mycoplasmal gene in malignant mammalian cells transformed by chronic persistent infection of mycoplasmas.

Chronic persistent infections by mycoplasmas induced malignant transformation of C3H mouse embryo cells that normally had never been reported to undergo spontaneous transformation. This mycoplasma-mediated oncogenic process had a long latency (more than 7 weeks of continuous mycoplasmal infection) and showed a multistage progression characterized by reversibility (at least up to 11 weeks of mycoplasmal infection) and irreversibility of malignant properties upon removal of the mycoplasma from culture. Further prolonged infections (18 weeks) by Mycoplasma fermentans or M. penetrans resulted in permanent transformation of these C3H cells that no longer required the continued presence of the transformation-inducing mycoplasmas in cultures to retain their malignant properties. Previous studies of viral oncogenesis revealed that virus-transformed cells always had viral gene(s) present. Integration of viral gene(s) apparently played an important role in the process of oncogenesis. In this study, we examined if the continued presence of any mycoplasmal gene(s) in mammalian cells, in whatever form, was also crucial in causing malignant cell transformation. Representational difference analysis (RDA) was a recently developed powerful technique to compare differences between two complex genomes. In the RDA system, subtractive and kinetic enrichment was used to purify and isolate restriction endonuclease gene fragment(s) of mycoplasmal origin, presumably present only in mycoplasma-transformed C3H cells, but not in nonmycoplasma-exposed control C3H cells. After three rounds of subtractive hybridization following PCR enrichment for each of three different restriction enzymes DNA digests, no gene fragment of mycoplasmal origin was amplified or identified in the permanently transformed C3H cells. Differing from tumorigenesis in animal cells induced by most oncogenic viruses or in plant cells induced by Agrobacteria, mycoplasmas evidently did not cause malignant transformation by integrating their gene(s) into the mammalian cell genome.

Isolation and Characterization of a Novel Gene Up-Regulated by Granulocyte Colony-Stimulation Factor in a Murine Progenitor Cell Line • 1409

Hematopoietic stem cells give rise to progenitor cells with progressively more restricted fates. This process is directed, in part, by a network of stimulating factors. Elucidation of the genetic events of hematopoiesis will provide insight into diseases of this system and novel strategies that may be employed to manipulate lineage commitment in a clinical setting. Granulocyte Colony-Stimulating Factor (G-CSF) is a hematopoietic growth factor which induces differentiation and maturation of the interleukin-3 (IL-3) dependent murine progenitor cell line 32D C13. In order to identify genes involved in commitment of myeloid progenitor cells to a differentiation program, 32D cells were withdrawn from IL-3 and cultured with G-CSF at a concentration of 400 U/ml. The cells were analyzed by napthol chloroesterase and myeloperoxidase staining at 24 hour time periods. At 96 hours after addition of G-CSF, 86% of the cells were napthol chloroesterase positive while 77% were myeloperoxidase positive. mRNA was extracted from a population of cells cultured for 96 hours with G-CSF, reverse transcribed to constitute the “tester” cDNA population, and used in a subtractive hybridization procedure with“driver” IL-3 cDNA. After two rounds of subtractive hybridization, the products were cloned into the Bluescript plasmid and screened for duplicates and true subtraction. DNA sequencing revealed the identity of a 164 base pair clone having 100% homology to the Tag-7 gene isolated in a differential display analysis of metastatic mouse tumors (Kustikova, et al; Genetika, 1996). Isolation of the full length cDNA sequence from a newborn murine brain library confirmed close homology to the Tag-7 gene. The deduced amino acid sequence encodes a novel protein that lacks known functional motifs. Both IL-3 and G-CSF induced 32D mRNA were probed for presence of the Tag-7 transcript by northern blot analysis. Signal quantitation showed a three-fold increase of Tag-7 message in the 96 hour G-CSF induced cells. Examination by RT-PCR indicated differential expression of Tag-7 in hematopoietic organs derived from adult and newborn mice. The authors conclude that the Tag-7 gene plays some yet undefined regulatory role in cell proliferation and differentiation. Further efforts are underway to isolate a corresponding human gene and to define the role of the Tag-7 gene in hematopoiesis.

Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.

Recovery of Probes Linked to thejcpkLocus on Mouse Chromosome 10 by the Use of an Improved Representational Difference Analysis Technique

Representational difference analysis (RDA) is a subtractive hybridization technique by which the differences between two complex genomes can be isolated. An improved version of this technique was used to isolate DNA segments that map to a narrow genetic region adjacent to thejcpklocus on Chromosome 10 of the mouse. A mutation at this locus acts recessively and causes an early onset polycystic kidney disease. Genomic subtractions involving DNA from C57BL/6 (B6) and its partially congenic partner,B6-jcpk/jcpk,produced 39 restriction fragments (difference products), 25 of which were unique and represented differences inBglII sites between these two strains. Although none identified thejcpklocus itself, 7 of these were mapped to an interval between 3.4 and 6.5 cM distal to thejcpklocus. Five of these 7 difference products were developed by subtracting B6-jcpk/jcpkfrom B6 DNA, but only 1 of the 5 was isolated using the original RDA technique. The other 4 were obtained by an improved technique that included size selection of difference products after the third round of subtractive hybridization and amplification. The remaining 2 of the mapped products resulted from the reciprocal subtraction experiment using the improvements. Thus, by this improved technique and two-way subtraction, we were able to add seven new markers to a relatively small genetic region on Chromosome 10.

Effective Production of RFLP Markers between Closely Related Varieties of Rice by Genomic Subtraction.

Polymorphic markers between closely related varieties of rice are necessary for the practical application of. molecular markers, such as restriction fragment length polymorphism (RFLP) markers, to rice breeding. In this study, we employed a representational difference analysis (RDA) to develop RFLP markers between closely related japonica varieties, Koshihikari and Akihikari. Three rounds of subtractive hybridization were performed, resulting in the isolation of 13 independent clones which showed RFLP between Koshihikari and Akihikari. These probes were unique and useful for mapping. Segregation of these markers in an F2 population derived from the cross between 2 varieties fitted the Mendelian expected ratio well, indicating that these clones were derived from the nuclear genome. Linkage analysis showed that these markers were separated into 2 groups and 5 unlinked markers. This is the first time that RDA was applied

Identification of enriched sequences from a cDNA subtraction-hybridization procedure.

The subtractive-hybridization procedure (see e.g. (1)) is a powerful procedure for obtaining cDNA populations which are enriched for upand down-regulated gene products. The method is based on the elimination of the common fragments within the cDNA populations being compared and is straight forward (1). Unfortunately, the analysis of the outcome is based on a screening hybridization assay and, hence, is indirect and time-consuming since individual molecules have to be cloned and analyzed. Another useful procedure for the isolation of specific gene products is the differential display method as developed by Liang and Pardee (2), recently optimized (3) and automated (4). In this procedure differential gene expression, in cells or tissues, is visualized by differences in the intensities of cDNA fragments after electrophoretic separation on a sequencing gel. After conversion into a cDNA using reverse transcriptase all gene products can be amplified as distinct, primer-dependent groups of cDNA by choosing a distinct oligo-dT-NN primer and a set of (short) random primers which facilitates identification (2). Although this procedure allows in principle for the identification of all mRNAs it can not be used for the isolation of underrepresented gene products. Here we report that the combination of both procedures, i.e. enriching a cDNA population by the subtractive-hybridization method (1) and identifying the enriched fragments by the differential display method (2) on a polyacrylamide gel yields a very powerful procedure that allows the identification of cDNAs that are enriched as little as 3-fold in the test fraction. In our laboratory we are studying the altered gene expression in a rat liver after a 70% hepatectomy. cDNA populations have been prepared from a normal and a regenerating (partially hepatectomized) rat liver (5). The subtractive-hybridization method as described by Wang and Brown (1), with minor modifications, was used to obtain the upand down-regulated gene products. Both cDNA populations were tagged with their own unique set of primers/adapters, CTCTTGCTTGAATTCGGACTA-3' and AATTCAGGCCAAGTCGGCCGG-3' for the normal and regenerating liver cDNA, respectively. In this way the necessity of linker removal, as described in the original procedure (1), at any point during the method was eliminated. The cDNA needed for each round of subtraction was amplified with Taq DNA polymerase (94°C for 1 min, 58°C for 90 s, and 72°C for 2 min with 30 cycles, followed by a final 5-min elongation-step at 72°C). The respective primers could not be interchanged since no product was detectable after a complete amplification cycle with a mismatched primer (not shown). In order to identify the upand down-regulated gene products after a completed subtractive-hybridization procedure, we adapted the display method from Liang and Pardee (2) with modifications. The two cDNA populations obtained after each round of subtractive-hybridization were amplified using Taq polymerase and the distinct primers in the presence of 0.5 jtM [a-S]dATP (1200 Ci/mmol) (final volume: 25 /xl). The experimental conditions were exactly the same as those used to generate the