The germline gene encoding DNA polymerase
[alpha]
in the hypotrichous ciliate
Oxytricha nova
is extremely scrambled
The germline gene encoding DNA polymerase [alpha] in the hypotrichous ciliate Oxytricha nova is extremely scrambled
David C.
Hoffman
and
David M.
Prescott*
Department of Molecular, Cellular and Developmental Biology, University of
Colorado,
Boulder
, CO 80309-0347,
USA
Received May 30, 1996
;
Revised and Accepted July 16, 1996
GenBank accession no. U58368
ABSTRACT
We report the structure of the micronuclear (germline) gene encoding the large
catalytic subunit of DNA polymerase
[alpha]
(DNA pol
[alpha]
) in the ciliate
Oxytricha nova
. It contains 44 internal eliminated segments (IESs) that divide the gene into
45 macronuclear-destined segments (MDSs) that are in a non-randomly scrambled order with an inversion near the gene center. Odd
numbered MDSs 29-43, containing 230 bp out of a total of 4938 bp of macronuclear sequence,
are missing from the 14 kb cloned gene. The missing MDSs have not been located
but are at least several kilobases from the main body of the gene. The
remarkably scrambled DNA pol [alpha]
gene must be extensively cut, re-ordered and spliced and an inversion must occur to produce an unscrambled,
functional version of the gene during development of a new macronucleus.
Unscrambling is hypothesized to occur by a homologous recombination mechanism
guided by repeat sequences at MDS ends.
INTRODUCTION
Genes in the germline micronuclear genome in hypotrichous ciliates are
interrupted by multiple non-coding DNA segments called internal eliminated segments (IESs) (
1
). The micronuclear gene segments separated by IESs are called macronuclear-destined segments (MDSs), which are spliced when IESs are excised during
development of a somatic macronucleus from a micronucleus after cell mating. In
some micronuclear genes MDSs are in a scrambled order and are spliced in the
correct order during macronuclear development (
2
,
3
). The analysis of a long, highly scrambled gene, encoding the large, catalytic
subunit of DNA polymerase [alpha] (DNA pol [alpha]) is reported here.
MATERIALS AND METHODS
Oxytricha nova
was grown and harvested and DNA prepared as described elsewhere (
3
). A single purified clone was selected from a [lambda] library (
1
) and amplified and purified according to established protocols (
4
). The insert was digested into three
Sac
I fragments, which were subcloned into the plasmid vector pGEM-7zf+ (Promega, Madison, WI). Sequencing was by the dideoxynucleotide chain
termination method using Sequenase (Amersham Life Sciences) and [[alpha]-
35
S]dATP (Dupont/New England Nuclear) incorporation.
RESULTS
A 14 kb micronuclear DNA fragment encoding DNA pol [alpha] was selected from a [lambda] genomic library of
O
.
nova
micronuclear DNA using fragments of the macronuclear DNA pol [alpha] gene (
5
) as selection probes. The probes were a 483 bp fragment from the 5'-end of the macronuclear gene, a fragment of 852 bp from the open
reading frame (ORF) and a
Sac
I fragment of 628 bp from the 3'-end of the macronuclear molecule. A purified [lambda] clone that hybridized with all three fragments was
digested with
Sac
I, yielding fragments of ~6.0, ~6.7 and ~1.3 kb. All three were subcloned into the plasmid pGEM-7zf+. The ~6.0 and ~6.7 kb inserts contained 5999 bp of the DNA pol [alpha] gene, including IESs, the 5' leader and 3' trailer and putative
flanking sequences of 776 bp at one end and 5963 bp at the other. The ~1.3 kb fragment contained additional flanking sequence of 1265 bp extending
from the 776 bp of flanking sequence.
The macronuclear DNA pol [alpha] gene sized molecule is 4938 bp long (
5
). Comparison of the macronuclear gene sized molecule with the micronuclear
sequence revealed that the micronuclear DNA pol [alpha] gene is divided into at least 45 MDSs separated by at least 44 IESs.
Numbers were assigned to MDSs according to the orthodox order in which they
appear in the macronuclear gene. Thirty seven MDSs are present in the [lambda] clone, but eight MDSs containing 230 bp are absent. The 37 MDSs are in
the order shown in Figure
1
. The 13 MDSs at one end of the gene (MDS 27 and even numbered MDSs 26-4) are inverted with respect to the remaining 24. The MDSs missing from
the [lambda] clone are 29, 31, 33, 35, 37, 39, 41 and 43. Repeats of 6-16 bp are present at the ends of those MDSs that must be re-ordered (Table
1
) and repeats of 2-4 bp are present where MDSs are already in the correct order with respect
to one another and need only to be spliced, i.e. MDSs 1, 2 and 3; 26 and 27; 44
and 45 (Fig.
1
). None of the 230 bp sequence in the eight missing MDSs is present in the DNA
that flanks the ends of the cloned gene (flanking DNA of 2041 bp at one end and
5963 bp at the other end). Library screens and PCR with micronuclear DNA have
so far failed to detect the missing 230 bp elsewhere in the genome, suggesting
that the missing sequence may be broken up into many more than eight MDSs.
DISCUSSION
Of the seven micronuclear genes of
O.nova
that have been sequenced, R1 (
6
), C2 (
1
), the gene encoding [beta] telomere binding protein (
7
) and the gene encoding heat shock protein 70 (
8
) contain IESs but are non-scrambled. Three genes, encoding actin I (
3
), [alpha] telomere binding protein ([alpha]TP) (
2
) and DNA pol [alpha] are scrambled. For example, the micronuclear [alpha]TP gene consists of 14 MDSs in the order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. The DNA pol [alpha]
gene is much longer and its non-random scrambling pattern is more complex than the patterns in the other
two scrambled genes. Like the [alpha]TP gene (14 MDSs), most of its MDSs fall into odd and even numbered
series. However, ~1.9 kb of the DNA pol [alpha] gene are inverted relative to the rest of the gene. In this
characteristic it is different from the [alpha]TP gene, which has no inversion, but is similar to the actin I gene,
which has a single, inverted MDS, however, the scrambling pattern of the DNA
pol [alpha] gene is unlike the actin I gene, whose nine MDSs are randomly scrambled
in the order 3-4-6-5-7-9-2-1-8 (
3
). Thus, the three scrambled micronuclear genes characterized so far show three
different combinations of inversions and random/non-random scrambled patterns of MDSs.
The DNA pol [alpha] gene contains 12 domains of amino acid sequence conserved among a wide
variety of other organisms from bacteriophage to humans and yeast (
9
,
10
), but these domains do not correspond in any discernible way to individual MDSs
or groups of MDSs in the
O.nova
gene. In some cases, conserved domains are actually split between two or three
MDSs.
The 14 kb micronuclear DNA [lambda] clone contains 4708 of the 4938 bp in the macronuclear DNA pol [alpha] gene. Missing from the micronuclear clone are 230 bp of the ORF.
The simplest assumption is that these missing 230 bp are present in eight
missing MDSs, numbered 29, 31, 33, 35, 37, 39, 41 and 43, as depicted in Figure
2
. This MDS pattern would complement the MDS pattern 28-30-32-34-36-38-40-42-44-45 present in the
micronuclear clone. The 2041 bp and 5963 bp of the insert that flank the cloned
gene contain none of the missing MDS sequences. A logical possibility is that
the missing MDSs are located >2041 bp into the DNA flanking MDS 27 and the 2041
bp are part of a very long IES connecting MDS 27 with MDS 29, as shown in
Figure
2
.
ACKNOWLEDGEMENT
Supported by NSF grant MCB9506287 to DMP.
REFERENCES
1 Klobutcher,L.A., Jahn,C.L. and Prescott,D.M. (1984) Cell, 36, 1045-1055.MEDLINE Abstract
