Comprehensive Sequence Analyses of 5' Flanking Regionsof Primate

Yoshimi Toda

12

ytoda@sfc.keio.ac.jp
1
2
3Alu

Elements

Rintaro Saito

12

rsaito@sfc.keio.ac.jp

Masaru Tomita

13

mt@sfc.keio.ac.jp

Laboratory for Bioinformatics
Graduate School of Media and GovernanceFaculty of Environmental Information, Keio University, 5322 Endo, Fujisawa 2520816, Japan

Abstract

Retrotransposons have been generally known to integrate randomly into host genomes. Jurka(1997) [3], however, showed consensus sequence patterns at integration sites of certain mammalian
retrotransposons, and suggested involvement of sequence speci c enzymes that mediate integration.
We have conducted comprehensive sequence analyses of 5' anking regions of primate Alu elements.
In contrast to the small but clean data set Jurka (1997) [3] used, (1) larger number of samples wereused, (2) wider region of 5' end of Alu elements was analyzed, and (3) comparisons were made among
di erent subfamilies for comprehensive analyses in order to identify characteristic sequence pattern(s)preceding 5' end of Alu elements. The nucleotide occurrences at each position within 500 bases of 5'
end of Alus were counted to obtain pro les. Information content at each nucleotide position in thesame region was, then, computed. Distinctive di erence in the nucleotide composition and information
content values that divides the region into two was observed. The region between 020 and 5' end of Alu
elements is found to be highly adenine-rich and shows signi cantly higher information content values
compared to the rest of the region, implying the existence of certain characteristic sequence patternin this region. Also, younger subfamilies of Alu elements show higher information content values thanolder subfamilies. This implies that certain characteristic sequence pattern already existed in theregion between 020 and 5' end of Alu elements at the time of Alu integration, and accumulation ofmutation in the course of time resulted in the less distinctive sequence pattern in older sequences.
Frequencies of all possible triplets (total of 64) were measured in the same region in order to identify
characteristic sequence pattern(s). Observation that frequencies of triplets \aaa," \taa" and \tta" inthe 5' anking sequences were high is consistent with Jurka (1997) [3]. Frequencies of some othertriplets such as \gaa," \caa," \aac," \ctt," \gtt," \atg," etc. which do not comprise the primary
candidates for the nick site in Jurka (1997), also show signi cantly high frequencies.

1 Introduction
Retrotransposons are a type of repetitive elements commonly found in genomes of various organisms, yet
the general mechanism of their integration has not been completely understood (Rogers, 1985 [9]). The
general understanding has been that their integration into host genomes occurs at random. However,Jurka (1997) [3] demonstrated that there are possible consensus sequence patterns at integration sites ofcertain mammalian retrotransposons, which strongly suggests sequence-speci c enzymatic involvementthat mediates integration. In the study conducted by Jurka, approximately 344 human Alu sequences
and 56 rodent ID sequences that retain full length with identical anking repeats at both ends werecarefully selected.
We have conducted comprehensive analyses of 5' anking regions of nearly 30,000 primate Alu elements, in order to identify sequence pattern(s) and their location that may be characteristic to the region.
The following 3 aspects make our study distinct from Jurka's work: (1) a larger number of samples wereused; (2) wider region of 5' upstream of Alu elements was considered; and (3) each of 12 subfamilies wasindividually analyzed.
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repetitive sequences constitute an Alu-family of short interspersed nucleic elements (SINEs). They
are about 300 base pairs (bps) long and are xed in primate genomes (Weiner et al., 1986 [11]). Their
copy number in a human genome is estimated to be several hundred thousand to one million, which
accounts for approximately 5 to 10% of the entire human genome (Weiner et al., 1986 [11]; Okada, 1991
[6]; Okada, 1994 [7]). Structurally, an Alu sequence consists of 2 similar subunits, connected with an
adenine-rich (A-rich) linker, and a 3' poly-A tail. This unit is usually anked immediately by 5' end and3' end direct repeats (4-10 bps long on the average) (Rinehart et al., 1980 [8]; Daniels and Deininger,1985 [2]).
Alu elements are subclassi ed into three groups, namely, Old, Middle, and Young. They are furtherclassi ed into 12 subfamilies (Old: Jo, Jb; Middle: Sz, Sx, Sq, Sp, Sg, Sc; Young: Y, Ya1, Ya5, Yb8) inaccordance with their putative time of proliferation and based on diagnostic positions in their nucleotide
sequences (Batzer et al., 1996 [1]).
In the current study, the nucleotide occurrences at each position in the 5' anking region of Alu
elements were counted to obtain pro les of nucleotide composition. We then computed information
content value (Schneider et al., 1986 [10]) at each nucleotide position to visualize possible locations of thecharacteristic nucleotide pattern. In order to specify the sequence pattern(s), frequencies of all possible
triplets (total of 64) were also measured.
Alu

2 Materials and Methods
2.1 Data
Primate Alu sequences longer than 250 bps were extracted from NCBI GenBank le release 103.0, alongwith their 5' anking sequences up to 500 bps long, a computer program named CENSOR (Jurka et al.
1996). Altogether, 29,663 Alu sequences were extracted and classi ed into 12 subfamilies. The numberof analyzed Alu sequences of each subfamily is shown in Table 1.

2.2 Nucleotide pro lesOccurrences of four nucleotides (adenine, thymine, guanine, cytosine) at each nucleotide position up
to 500 bps upstream of 5' anking region were counted in order to obtain nucleotide pro les for eachsubfamily.

2.3 Information content computation
The following formula was used to compute information content values which were standardized to avoid
in uence from base composition of analyzed sequence.
Information content = Oi log2 Oi =Ei(1)whereOi = Observed frequency of base i at speci c position
Ei = Expected frequency of base i at speci c position
An observed/expected (O/E) ratio of each nucleotide is de ned as the observed frequency divided by theexpected frequency, where the expected frequency is computed from the base composition of the wholeanalyzed sequences. High information content value at a certain position indicates a small variety in basecomposition, i.e., speci c nucleotide(s) appear at the position more often than expected.

422.4 Trinucleotide frequency
Frequency values of 64 possible trinucleotides were measured. The number of trinucleotides observed ata position was divided by the number of sequences analyzed and the resulted frequency was plotted foreach position.

3 Results and discussion
3.1 Pro le analysis
The nucleotide pro les of sequence composition upstream of 5' end of Alu elements show that the regionis A-T-rich. Particularly, A is the most prominent nucleotide especially in the region between 020 and
06 of 5' end of Alu elements (Table 2). Younger subgroups of Alu elements show more distinct A-richness.
Table 2(a) shows the nucleotide occurrences of the old subfamilies of Alu elements, Table 2(b) shows thatof the middle subfamilies, and Table 2(c) shows that of the young subfamilies. With all the subgroups,
the rate of A/T exceeds 60% at the positions 020 to 01. The rate of A exceeds 40% in the positions
between 016 and 06 with the oldest subgroup, the highest being 56.2%, between 017 and 06 with themiddle subgroup, the highest being 65.4%, between 017 and 05 with the youngest subgroup, the highest
being 67.1%. A-T-rich characteristics is as expected. It is known that Alu elements are inserted into
A-T-rich regions that may even include tails of pre-existing Alu elements (Rogers, 1985 [9]; Daniels andDeininger, 1985 [2]). The result also suggests that the insertion sites were more A-rich originally at thetime of insertion and mutation resulted in slightly smaller occurrences of A at the region which supports
Daniels and Deininger (1985) [2].

3.2 Information content
The existence of a characteristic nucleotide pattern was made more apparent from visualization by plotting the information content values onto a graph. In all the analyzed 12 subfamilies, information content
is high at the positions between 020 to 015 upstream of the 5' end of Alu elements. As shown in Fig. 1,
the information content vAlue is the highest with the youngest subfamilies (average information content
values of Ya1, Ya5, and Yb8 indicated with \Young" in the graph). This is expected as Alus belonging tothese subfamilies are evolutionarily young, that is, relatively short period of time has passed since theirintegration into the host genomes. The subfamilies Jo and Jb, which are evolutionarily the oldest, show
lower information content values compared to the younger ones (indicated with \Old" in Fig. 1). It issuspected that not only the sequences of Alu themselves, but also anking regions of old Alu elements
have accumulated mutations in the course of time and lost their original sequence pattern(s) in the region.
However, in contrast to the region between 020 and 010, randomness of nucleotide composition observedin the region further upstream of 020 shows no di erences among di erent subfamilies. This di erence ofrandomness in sequence composition between the anking region and the region further upstream of it,
and among di erent subfamilies imply that the observed characteristic sequence pattern at the anking
region already existed at the time of each Alu integration and random mutation have accumulated sincethen in the course of time.
The observation that the region, between 020 to 010 preceding the 5' end of Alu elements, shows highinformation content values is consistent with the analysis in Jurka (1997) [3]. Jurka computed 2 valuesof the suspected consensus patterns for each position of 30 bp region preceding 5' end of Alu elements
and shows that the positions between 019 to 010 preceding the 5' end of Alu elements demonstratesigni cantly high 2 values. Consistent with their analysis, our result strongly suggests the existence ofa certain characteristic sequence pattern(s) around this region.

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3.3 Trinucleotide frequencies
In order to identify DNA sequence pattern(s) involved in the high information content, frequencies of 64
possible triplets were measured. Alu sequences and their 5' anking regions of old, middle and younggroups were separately analyzed.
Of all the 64 possible triplet patterns, the most statistically signi cant are \aaa," \taa," and \tta".
Graphical visualization of the results are shown in Fig. 2. Placing these three trinucleotides in orderappearing in the analyzed sequences shows \ttaaaa" as the most frequently appearing pattern at thepositions between 017 and 013. This is consistent with Jurka (1996 [4{5], 1997 [3]) suggesting that thehexamer \ttaaaa" is the most prominent characteristic sequence pattern.
In our analyses, other triplets such as \aag," \aga," \gaa," \acc," \caa," \aca," \aac," \ctt," \gtt,"\cat," \cta," \tct," \atg," etc. also show high standardized frequencies. While their standardized
frequencies are lower than that of \aaa," \taa," or \tta," they are still statistically signi cant. Further
investigation is required for those triplets that do not seem to be related to the primary candidates fornick site for Alu integration in Jurka (1997).

4 Conclusion
In this study, comprehensive analyses of patterns in large anking regions of 5' end of Alu elements wereperformed by (1) counting nucleotide occurrences, (2) computing information content, and (3) measuring frequencies of triplet nucleotide patterns. Pro les of nucleotide occurrences show that the anking
region of 5' end of Alu elements are rich in adenine which emphasizes the fact that Alu elements areinserted into A-T rich regions and particularly into A-rich regions (Daniels and Deininger, 1985 [2]). Itis also shown that Alus that belong to the younger subgroup are more A-rich than the older ones. Acharacteristic sequence pattern \ttaaaa" was found through the information content analysis in conjunction with trinucleotide frequency analysis. Change in information content values among evolutionarily
di erent subgroups of Alus together with A-richness in younger subgroups of Alus further emphasize thepossibility that the characteristic sequence pattern existed where Alus would be integrated. Thus asJurka (1997) [3] suggests, the pattern found to be most frequently appearing in this region can be theprimary candidate for the nick site which is involved in Alu integration into host genomes. Some patterns
which were found to be not consistent with Jurka (1997) [3] need further investigation and the anking
region of 3' end of Alu elements also need to be analyzed for more supporting evidence of the enzymatic
involvement for Alu integration.

Acknowledgement
We would like to thank Dr. Jerzy Jurka, Paul Klonowski, and Jolanta Walichiewicz at the Genetic
Information Research Institute, Palo Alto, CA, for data on Alu elements. This work is supported inpart by a Grant-in-Aid for Scienti c Research on Priority Areas \Genome Science" from The Ministryof Education, Science, Sports and Culture in Japan.

References[1] Batzer, M.A., Deininger, P.L., Hellmann-Blumberg, U., Jurka, J., Labuda, D., Rubin, C.M., Schmid,C.W., Zietkiewicz, E. and Zuckerkandl, E., Standardized nomenclature for Alu repeats, J. Mol. Evol.,42:3{6, 1996.
[2] Daniels, G.R. and Deininger, P.L., Integration site preference of the Alu family and similar repetitive
DNA sequences, Nucleic Acids Res., 13: 8939{8954, 1985.
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Table 1: The number of samples of each subfamily used in our study.

Figure 1: Information content of 5' anking region of Alu elements.
45

Table 2: Nucleotide occurrences at each nucleic position in the 5' upstream anking region of Alu elements.

(a) Old subgroup(b) Middle subgroup(c) Young subgroup46

(a) Frequencies of trinucleotide \tta."(b) Frequencies of trinucleotide \taa."(c) Frequencies of trinucleotide \aaa."
Figure 2.

47[3] Jurka, J., Sequence Patterns indicate an enzymatic involvement in integration of mammalian retroposons, Proc. Natl. Acad. Sci. USA, 94: 1872{1877, 1997.
[4] Jurka, J. and Klonowski, P., Integration of retroposable elements in mammals: Selection of target
sites, J. Mol. Evol., 43: 685{689, 1996.
[5] Jurka, J., Klonowski, P., Dagman, V., Pelton, P., CENSOR { a program for identi cation andelimination of repetitive elements from DNA sequences, Comput. Chem., 20: 119{122, 1996.
[6] Okada, N., SINEs: short interspersed repeated elements of the eukaryotic genome, TREE, 6: 358{361, 1991.
[7] Okada, N., Retroposons as time markers of evolution, (Toki no jihyou to shite no retroposon),Proteins, Nucleic Acids, and Enzymes (Tanpakushicu, kakusan , kouso), 39:2724{2735, 1994.
[8] Rinehart, F.P., Ritch, T.G., Deininger, P.L. and Schmid, C.W., Renaturation rate studies of asingle family of interspersed repeated DNA sequences in human deoxyribonucleic acid, Biochem.,
20: 3003{3010, 1980.
[9] Rogers, J.H., The origin of retroposons, Int. Rev. of Cytol., 93: 187{279, 1985.
[10] Schneider, T.D., Stormo, G.D., Gold, L., Ehrenfeucht, A., Information content of binding sites onnucleotide sequences, J. Mol. Biol. 188(3):415{431, 1986.
[11] Weiner, A.M., Deininger, P.L. and Efstratiadis, A., Nonviral retroposons: genes, pseudogenes, andtransposable elements generated by the reverse ow of genetic information, Annu. Rev. Biochem.,
55:631{661, 1986.

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