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Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

Salvemini M, D'Amato R, Petrella V, Ippolito D, Ventre G, Zhang Y, Saccone G - BMC Genet. (2014)

Bottom Line: The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal.We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination.The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

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Analysis of SSH-MOS subtraction efficiency. A) Cclap-ps male-specific transcript expression profile at early embryonic stages: first lane= DNA marker, 1=unfertilized eggs; 2=XX/XY 0-0.5 hour AEL embryos; 3=XX/XY 0.5-3 hours AEL embryos; 4= XX/XY 3-24 hours AEL embryos; 5=XX-only 0-0.5 hour AEL embryos; 6=XX-only 0.5-3 hours AEL embryos; 7= XX-only 3-24 hours AEL embryos. Negative control not shown. B) last lane=DNA marker, Cclap-ps transcript expression profile at adult stages: 1=XY adult males; 2=XX adult females; 3=negative control. C) first lane= DNA marker, The subtracted SSH and subtracted SSH-MOS secondary PCR products were amplified using primers for Y-linked Cclap-ps transcript. Aliquots of the samples were analyzed by gel electrophoresis after 18, 23, 28, and 33 cycles of PCR amplification.
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Figure 2: Analysis of SSH-MOS subtraction efficiency. A) Cclap-ps male-specific transcript expression profile at early embryonic stages: first lane= DNA marker, 1=unfertilized eggs; 2=XX/XY 0-0.5 hour AEL embryos; 3=XX/XY 0.5-3 hours AEL embryos; 4= XX/XY 3-24 hours AEL embryos; 5=XX-only 0-0.5 hour AEL embryos; 6=XX-only 0.5-3 hours AEL embryos; 7= XX-only 3-24 hours AEL embryos. Negative control not shown. B) last lane=DNA marker, Cclap-ps transcript expression profile at adult stages: 1=XY adult males; 2=XX adult females; 3=negative control. C) first lane= DNA marker, The subtracted SSH and subtracted SSH-MOS secondary PCR products were amplified using primers for Y-linked Cclap-ps transcript. Aliquots of the samples were analyzed by gel electrophoresis after 18, 23, 28, and 33 cycles of PCR amplification.

Mentions: One of the major drawbacks of the SSH subtraction method is the high number of false positive clones, namely non-differentially expressed (redundant) cDNA species. These undesired background cDNA clones are generated from non-specific annealing of PCR primers or non-ligated adaptors (type-I background) and from redundant cDNA molecules that evade elimination by hybridization (type-II background). In order to reduce the number of background cDNA clones and the complexity of the cDNA mixture we applied the Mirror Oriented Selection (MOS) procedure [34]. MOS utilizes the principle that background molecules have only one orientation of the adaptor sequence, whereas truly differentially expressed molecules have many "progenitors" with adaptor sequences present in both orientations. The result is achieved by removal of one adaptor by restriction digestion, heat denaturation and re-annealing of the resulting cDNA molecules. In this case only hybrid molecules with the adaptors at the opposite ends are exponentially amplified by PCR, while background cDNAs by a linear PCR amplification. We partially validated the MOS procedure, using as reference a very early-transcribed (30' AEL, data not shown) Y-linked Cclap pseudo-gene serendipitously discovered in previous studies (Cclap-ps, unpub. res.; see Methods) which we expect would be enriched in the forward subtracted library (Figure 2 A-B).


Subtractive and differential hybridization molecular analyses of Ceratitis capitata XX/XY versus XX embryos to search for male-specific early transcribed genes.

Salvemini M, D'Amato R, Petrella V, Ippolito D, Ventre G, Zhang Y, Saccone G - BMC Genet. (2014)

Analysis of SSH-MOS subtraction efficiency. A) Cclap-ps male-specific transcript expression profile at early embryonic stages: first lane= DNA marker, 1=unfertilized eggs; 2=XX/XY 0-0.5 hour AEL embryos; 3=XX/XY 0.5-3 hours AEL embryos; 4= XX/XY 3-24 hours AEL embryos; 5=XX-only 0-0.5 hour AEL embryos; 6=XX-only 0.5-3 hours AEL embryos; 7= XX-only 3-24 hours AEL embryos. Negative control not shown. B) last lane=DNA marker, Cclap-ps transcript expression profile at adult stages: 1=XY adult males; 2=XX adult females; 3=negative control. C) first lane= DNA marker, The subtracted SSH and subtracted SSH-MOS secondary PCR products were amplified using primers for Y-linked Cclap-ps transcript. Aliquots of the samples were analyzed by gel electrophoresis after 18, 23, 28, and 33 cycles of PCR amplification.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4255797&req=5

Figure 2: Analysis of SSH-MOS subtraction efficiency. A) Cclap-ps male-specific transcript expression profile at early embryonic stages: first lane= DNA marker, 1=unfertilized eggs; 2=XX/XY 0-0.5 hour AEL embryos; 3=XX/XY 0.5-3 hours AEL embryos; 4= XX/XY 3-24 hours AEL embryos; 5=XX-only 0-0.5 hour AEL embryos; 6=XX-only 0.5-3 hours AEL embryos; 7= XX-only 3-24 hours AEL embryos. Negative control not shown. B) last lane=DNA marker, Cclap-ps transcript expression profile at adult stages: 1=XY adult males; 2=XX adult females; 3=negative control. C) first lane= DNA marker, The subtracted SSH and subtracted SSH-MOS secondary PCR products were amplified using primers for Y-linked Cclap-ps transcript. Aliquots of the samples were analyzed by gel electrophoresis after 18, 23, 28, and 33 cycles of PCR amplification.
Mentions: One of the major drawbacks of the SSH subtraction method is the high number of false positive clones, namely non-differentially expressed (redundant) cDNA species. These undesired background cDNA clones are generated from non-specific annealing of PCR primers or non-ligated adaptors (type-I background) and from redundant cDNA molecules that evade elimination by hybridization (type-II background). In order to reduce the number of background cDNA clones and the complexity of the cDNA mixture we applied the Mirror Oriented Selection (MOS) procedure [34]. MOS utilizes the principle that background molecules have only one orientation of the adaptor sequence, whereas truly differentially expressed molecules have many "progenitors" with adaptor sequences present in both orientations. The result is achieved by removal of one adaptor by restriction digestion, heat denaturation and re-annealing of the resulting cDNA molecules. In this case only hybrid molecules with the adaptors at the opposite ends are exponentially amplified by PCR, while background cDNAs by a linear PCR amplification. We partially validated the MOS procedure, using as reference a very early-transcribed (30' AEL, data not shown) Y-linked Cclap pseudo-gene serendipitously discovered in previous studies (Cclap-ps, unpub. res.; see Methods) which we expect would be enriched in the forward subtracted library (Figure 2 A-B).

Bottom Line: The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal.We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination.The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

View Article: PubMed Central - HTML - PubMed

ABSTRACT
The agricultural pest Ceratitis capitata, also known as the Mediterranean fruit fly or Medfly, is a fruit crop pest of very high economic relevance in different continents. The strategy to separate Ceratitis males from females (sexing) in mass rearing facilities is a useful step before the sterilization and release of male-only flies in Sterile Insect Technique control programs (SIT). The identification of genes having early embryonic male-specific expression, including Y-linked genes, such as the Maleness factor, could help to design novel and improved methods of sexing in combination with transgenesis, aiming to confer conditional female-specific lethality or female-to-male sexual reversal. We used a combination of Suppression Subtractive Hybrydization (SSH), Mirror Orientation Selection (MOS) anddifferential screening hybridization (DSH) techniques to approach the problem of isolating corresponding mRNAs expressed in XX/XY embryos versus XX-only embryos during a narrow developmental window (8-10 hours after egg laying, AEL ). Here we describe a novel strategy we have conceived to obtain relatively large amounts of XX-only embryos staged at 8-10 h AEL and so to extract few micrograms of polyA+ required to apply the complex technical procedure. The combination of these 3 techniques led to the identification of a Y-linked putative gene, CcGm2, sharing high sequence identity to a paralogous gene, CcGm1, localized either on an autosome or on the X chromosome. We propose that CcGm2 is a first interesting putative Y-linked gene which could play a role in sex determination. The function exterted by this gene should be investigated by novel genetic tools, such as CRISPR-CAS9, which will permit to target only the Y-linked paralogue, avoiding to interfere with the autosomal or X-linked paralogue function.

Show MeSH
Related in: MedlinePlus