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Nucleic Acids Research, 2000, Vol. 28, No. 23 4679-4688
© 2000 Oxford University Press

A unique pattern of intrastrand anomalies in base composition of the DNA in hypotrichs

David M. Prescott* and Sarah J. Dizick

University of Colorado, Department of Molecular, Cellular and Developmental Biology, Boulder, CO 80309-0347, USA

Received August 16, 2000; Revised and Accepted October 4, 2000.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The 50 non-coding bases immediately internal to the telomeric repeats in the two 5' ends of macronuclear DNA molecules of a group of hypotrichous ciliates are anomalous in composition, consisting of 61% purines and 39% pyrimidines, A>T (ratio of 44:32), and G>C (ratio of 17:7). These ratio imbalances violate parity rule 2, according to which A should equal T and G should equal C within a DNA strand and therefore pyrimidines should equal purines. The purine-rich and base ratio imbalances are in marked contrast to the rest of the non-coding parts of the molecules, which have the theoretically expected purine content of 50%, with A = T and G = C. The ORFs contain an average of 52% purines as a result of bias in codon usage. The 50 bases that flank the 5' ends of macronuclear sequences in micronuclear DNA (12 cases) consist of ~50% purines. Thus, the 50 bases in the 5' ends of macronuclear sequences in micronuclear DNA are islands of purine richness in which A>T and G>C. These islands may serve as signals for the excision of macronuclear molecules during macronuclear development. We have found no published reports of coding or non-coding native DNA with such anomalous base composition.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The DNA in the macronuclei of hypotrichs is organized in short molecules with lengths ranging from ~400 to ~15 000 bp with averages of ~2000 bp. With rare exceptions each molecule encodes a single gene. These short DNA molecules are derived from very high molecular weight DNA of micronuclear chromosomes after cell mating through a complex series of DNA processing steps that produce a new macronucleus. The first step in conversion of a micronucleus to a macronucleus is polytenization of the micronuclear chromosomes. In the polytene chromosome stage, thousands of short, non-coding segments called internal eliminated segments, or IESs, are eliminated from the genes that subsequently become the short macronuclear DNA molecules (1). The segments within a gene that are separated by IESs are called macronuclear-destined segments, or MDSs; these are ligated when IESs are removed. In some micronuclear precursors of macronuclear molecules the MDSs are in scrambled configuration. These MDSs become unscrambled and ligated in the orthodox order in conjunction with IES removal from polytene chromosomes. The polytene chromosomes are then fragmented, and ~95% or more of the original micronuclear DNA sequence complexity is eliminated. These eliminated sequences are the spacers in micronuclear DNA between the successive genes. The ~5% of sequence complexity that remains forms the gene-size molecules of the macronuclear genome. Telomeric repeats are added to the ends of the thousands of gene-size molecules as they are released from micronuclear DNA. Finally, the short molecules are replication-amplified to one to several thousand copies each, depending on the species.

The macronuclear, gene-size DNA molecules created by these processing steps generally consist of a single open reading frame (ORF) and 5' and 3' non-coding segments, as illustrated in Figure 1a. The ends of the molecules are capped with telomeric repeats of G4T4/C4A4 with a 16-base, single-stranded 3' overhang of 5'-T4G4T4G4-3' (except Euplotes species, which lack the last two Gs at the 3' ends of both telomeres). The 5' non-translated leaders and 3' non-translated trailers are AT-rich in contrast to the lower AT content of ORFs. The base compositions of 5' leaders, ORFs and 3' trailers are here documented quantitatively in the analysis of a group of 72 macronuclear DNA molecules.




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  		Figure 1. (a) The average structure of the 46 macronuclear DNA molecules in Table 1 with identified coding functions. 5' non-coding leader = 290 bp; ORF = 1410 bp; 3' non-coding trailer = 201 bp. (b) The AT percentage along the length of the macronuclear DNA molecule that encodes ßTP in O.trifallax. Averages were determined for 50-base segments that successively overlapped by 25 bases from point to point. Data are from DuBois and Prescott (25).

 
The main point of this paper is a previously unreported structural feature of macronuclear DNA molecules: regular intrastrand anomalies in base composition of non-coding regions at the ends of the molecules. This anomalous base composition at the ends of macronuclear molecules raises three questions: (i) how are such anomalies in base composition created and maintained; (ii) how are the anomalies restricted to particular segments of macronuclear DNA molecules and their micronuclear precursors; and (iii) what is the structural/functional significance of the anomalous base composition?


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Forty-six macronuclear DNA molecules with known coding functions and 34 molecules with unidentified coding functions studied in this report are listed in Table 1 with the organisms of origin, molecule lengths, GenBank accession numbers and references. Some of the molecules listed as unpublished were sequenced directly from PCR products. For others, PCR products were cloned in plasmid Puc 19 and sequenced. PCR products that included terminal, telomere repeats were generated with telomere primers of 5'-C4A4C4A4C4NN-3', where NN stands for one of 16 possible dinucleotides. Telomere primers were paired with primers at internal locations in molecules. Some of the unpublished sequences in Table 1 were determined on randomly picked, macronuclear molecules of Oxytricha nova cloned in plasmid Puc 19 using M13 and M13R primers for sequencing. Molecules in Table 1 listed as unpublished were sequenced by the departmental sequencing facility. Sequences of macronuclear molecules from Euplotes species (Table 2) were all obtained from GenBank. Molecules were analyzed with MacVector sequence analysis software.


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  		Table 1. Forty-six sequenced macronuclear DNA molecules with known coding functions from Oxytrichia, Stylonychia, Histriculus and Halteria species and 26 sequenced DNA molecules with unidentified coding functions from Oxytrichia nova
 

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  		Table 2. Forty-one sequenced macronuclear DNA molecules from Euplotes species
 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Overall characteristics of molecules
The two groups of macronuclear molecules listed in Table 1 were analyzed. One group consists of 46 molecules from Oxytricha, Stylonychia, Histriculus and Halteria species with identified coding functions, determined in various laboratories (see accession and reference numbers in Table 1). The second group consists of 26 molecules from O.nova with, as yet, unidentified coding functions, sequenced by us. The group of 46 molecules with known coding functions has well identified ORFs, and thus, the lengths of the non-coding 5' leaders and 3' trailers are well defined. The lengths of the 5' leaders and 3' trailers in the 26 molecules with unidentified coding functions have not been established. For this reason, the two groups of molecules were initially analyzed separately for base compositions at their ends.

Halteria is traditionally considered to be an oligotrich, not a hypotrich. However, its macronuclear DNA is organized into the short, gene-size molecules characteristic of hypotrichs (56; D.M.Prescott, unpublished results), and it replicates this DNA by means of replication bands (D.M.Prescott, unpublished results). The sequence of the ssrDNA in our Halteria strain indicates that it is closely related to the group including Oxytricha/Stylonychia, etc.

The 46 molecules with identified coding function range in length from 821 to 5007 bp (excluding telomere sequences), with an average of 1901 bp (Fig. 1a). 5' non-coding leaders range from 72 to 2230 bp, average 290 bp; ORFs range from 248 to 4542 bp, average 1410 bp; and 3' non-coding trailers range from 91 to 994 bp, average 201 bp. The base composition along the lengths of the molecules varies in a regular pattern. The AT bp content is high in the 5' leaders, ranging from 59 to 84%, averaging 72 ± 6%. The AT bp content decreases to an average of 52 ± 7% in the ORFs, and rises again to 70 ± 5% in the 3' trailers. This is illustrated in Figure 1b for the molecule encoding ß telomere binding protein (ßTP gene) in Oxytricha trifallax. The 5' leader is 78% AT, the ORF is 51% AT and the 3' trailer is 73% AT. The ßTP gene contains an intron of 86 bp, which is evident in Figure 1b by its high AT content (79%); a high AT content is characteristic of introns in hypotrich genes.

The 26 molecules from O.nova with unidentified coding function (Table 1) range in length from 292 to 6004 bp, with an average of 1900 bp. ORFs that occupy >50% of the lengths of many of these molecules were identified by start and stop codons, using the NCBI ORF finder. However, these ORFs have not been confirmed by other means, e.g., detection of mRNAs, cDNAs or encoded proteins, or by the known bias in codon usage in hypotrichs, and are not recorded in this report. None of these putative ORFs encode amino acid sequences with significant identity with amino acid sequences in GenBank (BLAST search). Thus, the 26 molecules of unknown coding function could not be used with confidence to define the total length of 5' leaders and 3' trailers. However, these molecules were useful in analyzing the composition of the 50 bases in non-coding regions at the very ends of molecules, immediately adjacent to the telomeres, since it can be safely assumed the non-coding 5' leaders and 3' trailers are longer than 50 bp. For example, for the 16 genes of known coding function in O.nova in Table 1 the leaders and trailers have average lengths of 299 bp (range, 82 to 1153) and 228 bp (range, 91 to 446), respectively.

Forty-one molecules from Euplotes species (Table 2) serve as a comparison group. Although Euplotes is a hypotrich, it is very distantly related to the hypotrich group (including Halteria) in Table 1 according to the sequence of the ssrDNA gene (57). As in other hypotrich species the AT base pair content in leaders and trailers is high, averaging 78 and 74%, respectively. The AT content of ORFs averages 59%.

Base composition at the ends of macronuclear molecules
The average percentage of A+G in the first 80 bp in the 5' end of the sense strand of the non-coding leader, and the 5' end of the antisense strand in the 46 molecules of known coding function listed in Table 1 is shown in 10-base segments in Figure 2a and b. The average A+G content is high in the first 60–70 bases in the 5' end of the sense strand and the first ~50 bases of the 5' end of the antisense strand. The A+G percentage then declines to 53–54% by 80 bases and falls further to ~50% in the rest of the non-coding leader and trailer (Table 3). Thus, the 5' ends of both strands are similarly purine rich.



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  		Figure 2. Average percentage content of A+G in 10-base segments for the 80 bases (a) in the 5' end of the sense strand and (b) at the 5' end of the antisense strand of 46 macronuclear molecules with known coding function. (c) Average percentage content of A+G in 10-base segments for the 80 bases in the 5' ends of both strands combined for 26 macronuclear molecules with unidentified coding functions from O.nova. (d) Average percentage content of A+G for 5' ends in (a), (b) and (c) (144 ends).

 

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  		Table 3. Average percentage base composition of the sense strand (5'->3') of 46 macronuclear molecules with identified coding functions
 
The corresponding values for the combined 5' ends of the two strands in the 26 molecules with unidentified coding functions are similar to the values for the 46 molecules with identified coding functions (Fig. 2c). The data for the two strands of the 26 molecules are combined because the orientation of their ORFs are unknown, and therefore leader and trailer ends are not distinguishable. The A+G content in the 5' ends ranges from 58 to 63% in the 10-base segments up to 50 bases and declines to 54–55% between 50 and 80 bases.

The average A+G percentages in 10-base segments for all 144 5' ends of the two strands in the 72 molecules are combined in Figure 2d. Average A+G percentages range from 58 to 63% in the 50 bases at 5' ends and decline to 54–56% between 50 and 80 bases. Although the averages for both groups of molecules in Table 1 show a dip in A+G content at ~15 bases (Fig. 2a, b and c), the dip is probably not significant because some individual molecules in both groups do not show the dip, although these molecules have the same overall high percentage of A+G in their 5' ends as all other molecules.

The data for the 10-base segments are of limited use because averages for such small short segments have high standard deviations, but these averages define roughly the extent, i.e. ~50 bases, of a consistent purine richness in the 5' ends. Therefore, the average percentage of A+G in the entire 50 base segment in the 5' ends was determined and compared to the A+G percentage in other parts of the molecules. This was done separately for the two groups of molecules in Table 1 because the molecules with established ORFs have defined 5' leaders and 3' trailers. The average percentages of A, T, G, C and A+G for the various segments of the molecules with established ORFs are given in Table 3. The first 50 bases in the 5' end of the sense strand contain an average of 61% A+G (range, 54–80%). Allowing for a transition from higher to lower purine content between 50 and 60 bases, the average content of A+G of the 5' strand in the leaders from 60 bases to the start of the ORF is 51%. The average percentage of A+G then rises slightly to 52% in the ORFs, declines to 49% in the 3' trailer, excluding the last 60 bases, and is 38% in the last 50 bases before the telomere. The pattern of A+G percentages is reciprocal, beginning at the 5' end of the complementary strand with corresponding numbers 62, 51, 48, 49 and 39%. Thus, the 50 bases at the 5' end of the sense strand and the 50 bases at the end of the antisense strand have the same purine richness, i.e. the ends of the molecule are symmetrical.

Results of similar analysis of the 26 molecules of O.nova with undefined ORFs are given in Table 4. In this case the 5' end of the sense strand cannot be distinguished from the 5' end of the antisense strand because the ORFs are unidentified. The base compositions of the 50 bases at the 5' end of one strand and the 50 bases at the 3' end of the same strand from each of the 26 molecules were averaged. The 50 bases at the 5' end are high in A+G (61%), whereas at the 3' end they are low in A+G (40%). Thus, the 5' ends of both strands are high in A+G, similar to the 5' ends of the 46 molecules in Table 3. The combined data for all 72 molecules (144 5' ends) showing the high average content of A+G in the 5' ends are given in Table 5.


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  		Table 4. Average percentage base composition of the 50 bases in the 5' and 3' ends of one strand of each of the 26 macronuclear molecules with unidentified coding functions from O.nova
 

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  		Table 5. Average percentage base composition of the 144 5' ends of strands in the 72 macronuclear molecules listed in Table 1
 
The average intrastrand percentages of the 4 bases in 50 bases in the 5' ends for the two groups of molecules both individually and for the combined groups are also given in Tables 3–5. The average percentages of A, T, G and C in each table are remarkably consistent, with values of 44, 32, 17 and 7%, respectively, in every data set. Thus, the purine richness in the 5' ends is primarily due to the high content of A residues. In addition, the ratios of A:T and G:C are anomalously high, violating parity rule 2 (58,59). According to this rule, A should equal T and G should equal C within a strand. Parity rule 2 may be violated for DNA subjected to strand-specific selection pressure, e.g., codon or mutational bias; for example, differences in mutation rates between leading and lagging strand templates during replication (60). The observation A!=T and G!=C in the 5' ends is in sharp contrast with the approximate, theoretically expected average A:T ratio of 36:35 and average G:C ratio of 14:15 for the combined leaders and trailers of the sense strand, minus the 60 bases at each end in the case of the 40 molecules with identified ORFs in Table 3. Both A and G contribute to the purine enrichment; A is increased 22% and G is increased 17% (Table 3; both 5' ends combined).

The average percentage ratio of A:T and G:C for the ORFs is 30:24 and 22:24, respectively, and the ORFs contain an average of 52% purines (Table 3). The ratio of G:C is essentially 1:1, but the ratio of A:T is somewhat higher, i.e. 30:24. The slightly higher purine percentage of 52% and the attendant higher ratio of A:T reflects both amino acid content of the encoded proteins and bias in codon usage for particular amino acids. An extreme example is the ORF encoding a glutamine/asparagine-rich protein in O.trifallax (AF188/63 in Table 1). Ninety-six out of 320 amino acids in this protein are glutamine, encoded predominantly by CAA and TAA, and asparagine, encoded predominantly by AAT and AAC. Thirty-nine percent of the bases in this ORF are A and 18% are G, resulting in an unusually high purine content of 57%. Bias in codon usage is pronounced in hypotrichs (61). Among the 31 most commonly used codons (used for >=30% of the occurrences of a particular amino acid), the average ratio of A:T and G:C is 33:26 and 18:23, respectively, which account at least in part for the greater occurrence of A in the 40 ORFs (Table 3).

A similar analysis was done on the 41 macronuclear molecules for the Euplotes species listed in Table 2. The Euplotes molecules are on average shorter (1557 bp) than molecules from the other hypotrichs (~1900 bp). The 5' leaders and 3' trailers in Euplotes are also shorter; averages of 147 and 128 bp, respectively. Nineteen of the 41 molecules have 5' leaders of 60 bp or less (range, 32–1262 bp), and 10 of the 41 have 3' trailers of 60 bp or less (range, 26–477 bp). Therefore, the analysis was done using the 30 bases (rather than 50 bases) at the 5' ends of the molecules so that all molecules could be included without invading ORFs. ORFs range from 324 to 3585 bp, with an average of 1161 bp. The purine content of the terminal 30 bases is 50 ± 7%, significantly below the 61% found in the other hypotrich group. The intrastrand ratios of A:T and G:C for these 30 bases are exactly 1:1 for the two 5' ends combined (percentages of A, T, G and C of 38, 38, 12 and 12 respectively, Table 6). ORFs contain an average of 55% purines and average percentages for A, T, G and C of 34, 25, 21 and 20 respectively. A is higher than T, and total purine content is high; this is the result, at least in part, of codon bias. Thus, Euplotes molecules do not have the anomalous base composition in the 5' ends seen in the other hypotrichs.


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  		Table 6. Average percentage base composition of the 30 bases in the 5' ends of 41 macronuclear DNA molecules (82 total ends) from Euplotes species listed in Table 2
 
Sequences flanking genes in the micronuclear DNA
Within the micronuclear chromosomes, macronuclear sequences lack telomere repeats and are flanked by micronuclear-specific DNA that is eliminated during macronuclear development. The sequences of seven micronuclear-flanking DNA segments at the two ends of the macronuclear sequences encoding the scrambled {alpha} telomere protein ({alpha}TP) (62), the non-scrambled gene encoding hsp 70 (K.R.Lindauer, R.C.Anderson and D.M.Prescott, unpublished results), and the non-scrambled C2 gene (63) plus the sequence of the DNA flanking the 3' end of the scrambled DNA polymerase {alpha} (DNA pol {alpha}) gene (64) are available in O.nova. In the 14 MDSs of the scrambled {alpha}TP gene, MDSs 1 and 14 are in terminal positions, so that the first 50 bases of the 5' leader and the last 50 bases of the 3' trailer are in the orthodox positions in the micronuclear version of the gene. The same is true for the 3' end of the scrambled micronuclear DNA pol {alpha} gene, but the 5' end of this gene (MDS 1) is internal in the gene because of an inversion.

In the 50-base segment that flanks the 5' end of the sense or antisense strand (seven cases) the average percentages of A, T, G and C are 36, 41, 11 and 12 (Table 7). This composition differs markedly from that in the immediately adjacent 50 bases in the 5' ends of the macronuclear sequences (Table 3). For example, in the 50-base flanking segments the intrastrand A:T and G:C ratios are close to 1:1, and the average purine content is 47 ± 17% versus 61 ± 6% in the first 50 bases in the macronuclear sequence. Clearly, a transition takes place from orthodox A:T and G:C ratios (1:1) and orthodox percentage of A+G (47%) in the flanking strand to anomalous A:T and G:C ratios (44:32 and 17:7, respectively) and an anomalously high A+G percentage (61%) in the first 50 bases of the macronuclear sequence.


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  		Table 7. Average percentage base composition in the 50 bases in micronuclear DNA immediately flanking ends of 5' strands of seven macronuclear sequences (with telomeres) and five reconstructed flanking sequences for scrambled genes. The right column with the average percentage A+G for macronuclear sequences is included for comparison
 
According to the model for unscrambling of the DNA pol {alpha} gene in O.nova during macronuclear development (65,66), unscrambling leaves the gene still integrated in its micronuclear chromosome, and displaces IESs 13, 16, 12, 17 and 11 (total, 48 bases), arranged in that order, to the flank of MDS 1. According to the model, the gene is then excised from the chromosome by cutting between the flanking DNA formed by IESs 13, 16, 12, 17 and 11 and the end of MDS 1. Similarly, in the DNA pol {alpha} gene of O.trifallax the 5' flanking sequence is formed by IESs 15 and 19 as a result of unscrambling, according to the recombination model of unscrambling. Similarly, the flanking DNA at the 5' end of the sense strand of the actin I gene in O.nova is formed by IESs 7, 8 and 5 as a result of unscrambling. The flanking DNA at the 5' end of the actin I gene in O.trifallax is formed by IESs 8, 9 and 5. The flanking sequence at the 3' end of the sense strand of the actin I gene in O.nova is formed by IES 6. The average base composition for these five theoretically reconstituted flanking sequences is A = 36, T = 36, G = 12 and C = 16 (Table 7). In contrast, the first 50 bases in the 5' ends of the relevant macronuclear sequences contain an average 62 ± 17% A+G (Table 7). Thus, reconstructed flanking sequences for scrambled genes show the same transition at their junction with macronuclear sequences, after unscrambling, as do the orthodox flanking sequences. The combined averages for the seven orthodox and five reconstructed flanking sequences are 36:39 for A:T and 12:13 for G:C. The A+G percentages are 47 and 48%, respectively.

The picture that emerges for the micronuclear version of a macronuclear molecule is summarized in Figure 3. In flanking DNA reading 5'->3' in the sense strand, A+G = 47% and A {cong} T and G {cong} C. In the first 50 bases after the transition into the macronuclear sequence (leader or trailer), A+G = 61% and A>T and G>C. After the first 50 bases A+G is again ~50% and A {cong} T and G {cong} C in the rest of the leader and trailer. In the ORF A+G = 52% (A>T and G = C). Thus the average purine content changes from ~50% in flanking DNA to 61% in the first 50 bases to an average of 50% in the remainder of the leader and trailer and 52% in the ORF.



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  		Figure 3. Composite summary of the average A+G content and A:T and G:C ratios of 50 bases of micronuclear flanking sequences and of various parts of the macronuclear sequences.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Approximately 50 bp at the ends of macronuclear molecules from the Oxytricha/Stylonychia/Histriculus/Halteria group of ciliates is anomalous in its base composition. In the 5' ends of strands, A>T (ratio of 44:32) and G>C (ratio of 17:7), resulting in purine richness (purine:pyrimidine ratio of 61:39), primarily because of the high percentage of A residues (44%). An identical, average, anomalous composition in 5' ends (A = 44%, T = 32%, G = 17% and C = 7%) was observed for a group of 46 molecules with identified coding functions and for a group of 26 molecules of unidentified coding functions. The anomalous composition is in contrast to the rest of the non-coding leaders and trailers in the 46 molecules with identified coding functions, in which A {cong} T and G {cong} C, and A+G = 50%. The high purine content of the 50 bases in the ends of 5' strands also contrasts with the average composition of the 5' strands in ORFs, in which A is a little higher than T, and G {cong} C, and the total purine content is 52%. The slight average richness of A in ORFs is due to bias towards the use of A-rich codons in these ciliates and the amino acid composition of the proteins encoded by the 46 genes.

The anomalous base composition observed in the 5' ends of macronuclear molecules is present in the micronuclear DNA and is transmitted to macronuclear molecules during genome processing after cell mating. This suggests that the significance of the anomalous base composition resides in some function or activity in the micronuclear genome. Since the anomalous composition occurs in both 5' ends of macronuclear precursors in micronuclear DNA and, in some cases, is separated from ORFs by hundreds of base pairs, it is unlikely that significance of the anomalous composition is in any way connected to coding functions of genes.

Two possibilities are that the anomalous base composition derived from differential mutation rates in the two strands during DNA replication or originated by selection as a signal to mark the ends of micronuclear precursors of macronuclear molecules. For example, anomalous base composition in bacterial DNA has been suggested to arise from preferential deamination of bases in lagging strand templates, converting C->T and A->G, so that T>A and G>C in that strand (67). Lagging strand templates are transiently single-stranded, and single-stranded DNA is deaminated in vitro more than 100 times more rapidly than double-stranded DNA (68,69). How such a replication effect could be restricted to ~50 bases at the ends of micronuclear precursors of macronuclear molecules is problematic. In any case, the deamination idea would presumably create duplex DNA in which T>A and G>C in one strand (A>T and C>G in the complementary strand) but in macronuclear ends A>T and G>C in one strand, and T>A and C>G in the complement. Thus, deamination appears unlikely to be responsible for the anomalous base composition of macronuclear molecular ends.

The anomalous base composition in hypotrichs could possibly act as a signal for excision of macronuclear molecules from micronuclear DNA during macronuclear development after cell mating. One test of this hypothesis is the base composition of DNA that flanks macronuclear precursors in micronuclear DNA. In 50-base segments flanking the 5' end of the sense strand of three genes and 50-base segments flanking the complement to the 3' end of the sense strand of four genes, the average base A:T and G:C base ratio is 36:41 and 11:12, respectively, and the A+G average is 47% (Table 7). These are orthodox values and contrast with the anomalous base percentages in the 50 bases in the 5' ends of the immediately adjacent macronuclear sequences.

Five additional flanking sequences reconstructed by unscrambling the micronuclear precursors of actin I and DNA pol {alpha} molecules, using the recombination model, also have, on average, orthodox base compositions (Table 7). Thus, the 50 bp in the ends of micronuclear precursors are islands of anomalous base composition embedded in sequence of orthodox composition, i.e. A = T, G = C and A+G = ~50%. This is consistent with the idea that the anomalous base composition marks the ends of micronuclear precursors of macronuclear genes and might serve as identifying targets for excision of macronuclear molecules in these hypotrichs.

In contrast, the ends of 41 macronuclear molecules in Euplotes species do not have anomalous compositions. However, Euplotes molecules contain a 10-bp consensus sequence 17 bp downstream or upstream of apparent excision points that acts to identify those excision points (36). No consensus sequences have been detected in the 50-bp segments either in micronuclear precursors or in micronuclear flanking sequence in the group including Oxytricha/Stylonychia, etc. Both poly(A) and poly(T) tracts up to 6 bases long are present, but they occur at the expected frequency for AT-rich DNA and occupy random positions in the 50-base segments at 5' ends of strands.

Anomalous base compositions could possibly act as a target for proteins responsible for excision of macronuclear molecules. Such a target signal would presumably be imprecise since it is diffuse, and there is no discernible specific pattern in the sequences with the anomalous base composition. This imprecision is consistent with the earlier finding by Baird and Klobutcher (70) that in O.nova the telomere addition site (excision site) for a particular macronuclear molecule can vary over a range of several base pairs. This has been confirmed in several hypotrichs in the Oxytricha/Stylonychia group (K.E.Croft, K.E.Orr and D.M.Prescott, unpublished results). In contrast, the excision point in Euplotes, which has an apparent specific signal sequence, is precise to the base pair (70).

What structural property of the anomalous 50-bp segments might be specifically recognized by excision machinery is not known. Anomalous base composition might, however, affect DNA structure to produce a specific, recognizable target, perhaps by creating a differential chromatin structure along micronuclear DNA. Finally, we have been unable to find any published reports of native, non-coding DNA sequences with the extreme anomaly in base composition described here for 5' ends of macronuclear DNA.


   ACKNOWLEDGEMENTS
 
We thank Gayle Prescott for manuscript preparation and David Hoffman for technical assistance with cloning of some DNA molecules. This work is supported by NSF grant MCB-9974680 to D.M.P.


   FOOTNOTES
 
* To whom correspondence should be addressed. Tel: +1 303 492 8381; Fax: +1 303 492 7744; Email: prescotd{at}spot.colorado.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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