Limits...
The compositional landscape of minicircle sequences isolated from active lesions and scars of American cutaneous leishmaniasis.

Rodrigues EH, Soares FC, Werkhäuser RP, de Brito ME, Fernandes O, Abath FG, Brandão A - Parasit Vectors (2013)

Bottom Line: Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides.With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer.It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: American cutaneous leishmaniasis (ACL) is characterized by cutaneous lesions that heal spontaneously or after specific treatment. This paper reports on the analysis of kDNA minicircle sequences from clinical samples (typical lesions and scars) that were PCR-amplified with specific primers for Leishmania species of the subgenus Viannia.

Methods: From 56 clinical isolates we obtained a single amplified fragment (ca. 790 bp), which after cloning and sequencing resulted in 290 minicircle sequences from both active lesions and scars. We aimed to get a compositional profile of these sequences in clinical samples and evaluate the corresponding compositional changes. Sequences were analyzed with the compseq and wordcount (Emboss package) to get the composition of di-, tri-, tetra-, penta- and hexanucleotides. Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides.

Results: This compositional analysis showed that minicircles amplified from active cutaneous lesions and scars have a distinct compositional profile as viewed by nucleotide composition of words up to 10mer. With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer.

Conclusion: These results indicate that minicircle sequences can be monitored upon direct exposure to a selection/stressing environment (e.g. chemical action) by evaluating their nucleotide compositional profile. It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.

Show MeSH

Related in: MedlinePlus

Dinucleotide frequencies of minicircles from Leishmania (Viannia) braziliensis obtained from clinical samples of patients before and after treatment (typical lesions and scars, respectively) of the ACL. Human lesions (black bars) and scars (gray bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3750493&req=5

Figure 1: Dinucleotide frequencies of minicircles from Leishmania (Viannia) braziliensis obtained from clinical samples of patients before and after treatment (typical lesions and scars, respectively) of the ACL. Human lesions (black bars) and scars (gray bars).

Mentions: The most frequent dinucleotides were AA, TT, AT and TA, ranging from 10 to 18% in frequency. Figure 1 shows that dinucleotide frequencies are slightly higher for sequences obtained from scars. A Wilcoxon signed rank test points to no significant difference for dinucleotide frequencies from active lesions and scar sequences, but for words above 3mer this value drops significantly (Table 3). Thus, at the dinucleotide level we cannot detect differences in minicircle sequence composition due to chemical pressure induced by the drug treatment. As expected from minicircle heterogeneity, for words of 3-, 4-, 5-, and 6mer the variation in frequency starts to be significant between an active lesion and a scar. The comparative analysis of minicircles poses the question of how their widely known sequence heterogeneity should be evidenced when the whole sequences are fragmented into their basic words. These words apparently have specific information, which in the case of trypanosomatids might be viewed as a sort of minicircle signature. By adding the composition of trinucleotides up to hexanucleotides it is possible to show the appearance of compositional heterogeneity with direct comparison of frequencies in both set of sequences, lesions and scars. Figure 2 summarizes this comparison at nucleotide length at the range 2- to 6mer in clinical samples for both sets (human lesions and scars). The graphics show the frequency variation as compared word by word after sorting the frequencies in decreasing order (sorting was fixed for the active set of sequences). This direct comparison is grounded in the fact that if no bias exists between the two sets, all the frequencies would display a straight line. Of course, as minicircle sequences are both multi-copies and extensively variable, we do observe some level of heterogeneity regardless of whether the sequence is partial or full length. This analysis shows the bias increasing along with the word length. For example, above the tetranucleotide level specific words start to appear at higher frequencies in one of the two sets. Zero frequency words are not counted for this graphical display, but it might be possible to show the nucleotide bias by counting words that are exclusively absent in both sets. The frequency graphics also show that words longer than 4mer are more sensitive to small variations in minicircle composition. This is expected from such an analysis because as we progress towards longer words, a single mutation in only one set can create a new word. Thus, larger fluctuations in frequency are observed in the lower part of the graphics, as can be observed for tetra- and pentanucleotides. On the other hand, increasing nucleotide word length also uncovers the zero frequency words, which lowers the interval of frequency variation. That is the case for 6mer (Figure 2). These observations imply that in the range 4- to 6mer, most of the nucleotide heterogeneity is represented by words of very low frequency. Overall, the graphics point that nucleotide word frequency from scar sequences is slightly lower than in active lesions.


The compositional landscape of minicircle sequences isolated from active lesions and scars of American cutaneous leishmaniasis.

Rodrigues EH, Soares FC, Werkhäuser RP, de Brito ME, Fernandes O, Abath FG, Brandão A - Parasit Vectors (2013)

Dinucleotide frequencies of minicircles from Leishmania (Viannia) braziliensis obtained from clinical samples of patients before and after treatment (typical lesions and scars, respectively) of the ACL. Human lesions (black bars) and scars (gray bars).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750493&req=5

Figure 1: Dinucleotide frequencies of minicircles from Leishmania (Viannia) braziliensis obtained from clinical samples of patients before and after treatment (typical lesions and scars, respectively) of the ACL. Human lesions (black bars) and scars (gray bars).
Mentions: The most frequent dinucleotides were AA, TT, AT and TA, ranging from 10 to 18% in frequency. Figure 1 shows that dinucleotide frequencies are slightly higher for sequences obtained from scars. A Wilcoxon signed rank test points to no significant difference for dinucleotide frequencies from active lesions and scar sequences, but for words above 3mer this value drops significantly (Table 3). Thus, at the dinucleotide level we cannot detect differences in minicircle sequence composition due to chemical pressure induced by the drug treatment. As expected from minicircle heterogeneity, for words of 3-, 4-, 5-, and 6mer the variation in frequency starts to be significant between an active lesion and a scar. The comparative analysis of minicircles poses the question of how their widely known sequence heterogeneity should be evidenced when the whole sequences are fragmented into their basic words. These words apparently have specific information, which in the case of trypanosomatids might be viewed as a sort of minicircle signature. By adding the composition of trinucleotides up to hexanucleotides it is possible to show the appearance of compositional heterogeneity with direct comparison of frequencies in both set of sequences, lesions and scars. Figure 2 summarizes this comparison at nucleotide length at the range 2- to 6mer in clinical samples for both sets (human lesions and scars). The graphics show the frequency variation as compared word by word after sorting the frequencies in decreasing order (sorting was fixed for the active set of sequences). This direct comparison is grounded in the fact that if no bias exists between the two sets, all the frequencies would display a straight line. Of course, as minicircle sequences are both multi-copies and extensively variable, we do observe some level of heterogeneity regardless of whether the sequence is partial or full length. This analysis shows the bias increasing along with the word length. For example, above the tetranucleotide level specific words start to appear at higher frequencies in one of the two sets. Zero frequency words are not counted for this graphical display, but it might be possible to show the nucleotide bias by counting words that are exclusively absent in both sets. The frequency graphics also show that words longer than 4mer are more sensitive to small variations in minicircle composition. This is expected from such an analysis because as we progress towards longer words, a single mutation in only one set can create a new word. Thus, larger fluctuations in frequency are observed in the lower part of the graphics, as can be observed for tetra- and pentanucleotides. On the other hand, increasing nucleotide word length also uncovers the zero frequency words, which lowers the interval of frequency variation. That is the case for 6mer (Figure 2). These observations imply that in the range 4- to 6mer, most of the nucleotide heterogeneity is represented by words of very low frequency. Overall, the graphics point that nucleotide word frequency from scar sequences is slightly lower than in active lesions.

Bottom Line: Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides.With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer.It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Background: American cutaneous leishmaniasis (ACL) is characterized by cutaneous lesions that heal spontaneously or after specific treatment. This paper reports on the analysis of kDNA minicircle sequences from clinical samples (typical lesions and scars) that were PCR-amplified with specific primers for Leishmania species of the subgenus Viannia.

Methods: From 56 clinical isolates we obtained a single amplified fragment (ca. 790 bp), which after cloning and sequencing resulted in 290 minicircle sequences from both active lesions and scars. We aimed to get a compositional profile of these sequences in clinical samples and evaluate the corresponding compositional changes. Sequences were analyzed with the compseq and wordcount (Emboss package) to get the composition of di-, tri-, tetra-, penta- and hexanucleotides. Additionally, we built a nucleotide dictionary with words of 7, 8, 9 and 10 nucleotides.

Results: This compositional analysis showed that minicircles amplified from active cutaneous lesions and scars have a distinct compositional profile as viewed by nucleotide composition of words up to 10mer. With regard to the most frequent nucleotide words above length 6, there is also a distinct pattern for 7, 8, 9 and 10mer.

Conclusion: These results indicate that minicircle sequences can be monitored upon direct exposure to a selection/stressing environment (e.g. chemical action) by evaluating their nucleotide compositional profile. It might be useful as a molecular tool in research concerning the evolution of infecting Leishmania in both vector and vertebrate hosts.

Show MeSH
Related in: MedlinePlus