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A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus

Midgut degradation of inhibitor.Amplification of 18s sequences from fish in the presence of foregut (FG) extracts and FG mixed with midgut (MG) suspensions from the same individuals (N = 3). Bands of PCR product indicate the neutralization of the foregut inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled.
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fig-5: Midgut degradation of inhibitor.Amplification of 18s sequences from fish in the presence of foregut (FG) extracts and FG mixed with midgut (MG) suspensions from the same individuals (N = 3). Bands of PCR product indicate the neutralization of the foregut inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled.

Mentions: Amplification of control DNA with 18s in the presence of foregut extracts was possible if midgut suspensions were present in the mix, thus suggesting regulation of the inhibitor in the midgut (Fig. 5). The effect of the midgut component (or components) counteracting the activity of the inhibitor was detected even at 10−4 dilutions of the original midgut aliquot (Fig. 6). For the sample FG3 + MG3(10−4) there was only a faint band, suggesting that the dilution of the midgut was approaching the limit of activity against the inhibitor. For these two experiments, both positive and negative controls showed the expected patterns (Figs. 5 and 6).


A localized PCR inhibitor in a porcelain crab suggests a protective role.

El-Maklizi MA, Ouf A, Ferreira A, Hedar S, Cruz-Rivera E - PeerJ (2014)

Midgut degradation of inhibitor.Amplification of 18s sequences from fish in the presence of foregut (FG) extracts and FG mixed with midgut (MG) suspensions from the same individuals (N = 3). Bands of PCR product indicate the neutralization of the foregut inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260131&req=5

fig-5: Midgut degradation of inhibitor.Amplification of 18s sequences from fish in the presence of foregut (FG) extracts and FG mixed with midgut (MG) suspensions from the same individuals (N = 3). Bands of PCR product indicate the neutralization of the foregut inhibitor. Positive and negative controls are as in Fig. 4. Empty lanes are not labeled.
Mentions: Amplification of control DNA with 18s in the presence of foregut extracts was possible if midgut suspensions were present in the mix, thus suggesting regulation of the inhibitor in the midgut (Fig. 5). The effect of the midgut component (or components) counteracting the activity of the inhibitor was detected even at 10−4 dilutions of the original midgut aliquot (Fig. 6). For the sample FG3 + MG3(10−4) there was only a faint band, suggesting that the dilution of the midgut was approaching the limit of activity against the inhibitor. For these two experiments, both positive and negative controls showed the expected patterns (Figs. 5 and 6).

Bottom Line: By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure.Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound.The identity of the inhibitory molecule remains unknown.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biology Department, The American University in Cairo , New Cairo , Egypt.

ABSTRACT
A number of polymerase chain reaction (PCR) inhibitors have been identified from biological and environmental samples. By and large, such substances are treated as random nuisances and contaminants with alternate functions; their inhibitory effects on DNA replication being a coincidental property of their molecular structure. Here, we demonstrate the presence of a localized PCR inhibitor in the foregut of the porcelain crab Petrolisthes rufescens (Anomura: Porcellanidae) from the Red Sea. The inhibitor precluded amplification of 28s, 16s and 18s gene sequences effectively but lost activity at 10(-2) dilutions from initial concentration. Heat treatment was ineffective in arresting inhibition and spectrophotometric techniques suggested that the inhibitor was not a melanin-type compound. The compound was not detected from midgut, hindgut, or gills of the crab. Activity of the inhibitor was precluded when samples were treated with suspensions from the midgut, suggesting that enzymatic degradation of the inhibitor likely happens at that part of the gut. As many microbial pathogens invade their hosts via ingestion, we suggest the presence of the localized inhibitor could carry a defensive or immunological role for P. rufescens. The identity of the inhibitory molecule remains unknown.

No MeSH data available.


Related in: MedlinePlus