Download An archaebacterial homolog of pelota, a meiotic cell division protein

MICROBIOLOGY
LETTERS
ELSEVIER
FEMS Microbiology Letters 144 (1996) 151-155
An archaebacterial homolog of pelota, a meiotic
cell division protein in eukaryotes
Mark A. Ragan ajbl*, John M. Logsdon Jr. c, Christoph W. Sensen alb,
Robert L. Charlebois a,d, W. Ford Doolittle asc
’ Program in Evolutionary Biology, Canadian Institute for Advanced Research, National Research Council of Canada, 1411 Oxford Street,
Halifax, Nova Scotia B3H 321. Canada
b Institute for Marine Biosciences, National Research Council of Canada, 1411 Oxford Street, Halifax, Nova Scotia B3H 321, Canada
’ Department of Biochemistry, Sir Charles Tupper Medical Building, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada
’ Department of Biology, University of Ottawa, 30 Marie Curie, Ottawa, Ontario KlN 6N5, Canada
Received8 August 1996; revised 28 August 1996; accepted29 August 1996
Abstract
An open reading frame (pelA) specifying a homolog of pelota and DOM34, proteins required for meiotic cell division in
melanogaster and Saccharomyces
cerevisiae, respectively, has been cloned, sequenced and identified from the
archaebacterium Sulfolobus solfataricus. The S. solfataricus PelA protein is about 20% identical with pelota, DOM34 and the
hypothetical protein R74.6 of Caenorhabditis elegans. The presence of a pelota homolog in archaebacteria implies that the
meiotic functions of the eukaryotic protein were co-opted from, or added to, other functions existing before the emergence of
eukaryotes. The nuclear localization signal and negatively charged carboxy-terminus characteristic of eukaryotic pelota-like
proteins are absent from the S. solfataricus homolog, and hence may be indicative of the acquired eukaryotic function(s).
Drosophila
Keywords: (Sulfolobus solfataricus) ;
Archaea; Archaebacterium; Meiosis; Pelota; DOM34
1. Introduction
Archaebacteria
(Archaea) are phylogenetically
distinct from eubacteria (Bacteria) and eukaryotes (Eukarya) [l-4]. Phylogenetic analyses of macromolecular sequences suggest that within the archaebacteria
there are two main lineages, Euryarchaeota
(methanogens, halobacteria
and some thermophiles)
and
Crenarchaeota
(other thermophiles, including Su2folobus) [l]. Although most molecular-sequence
anal* Corresponding author. Tel. : +l (902) 426-1674;
Fax: +l (902)-426-9413;E-mail: [email protected]
0378-1097 /96/$12.00 Copyright
PIISO378-1097(96)00351-5
0 1996 Federation
of European
yses indicate that the Archaebacteria
are monophyletic [2,3], there is some evidence that they may
be paraphyletic [4], with the crenarchaeotes
as the
sister-group of eukaryotes. In either case, archaebacteria and eukaryotes are thought to share a common
ancestor more recent than that shared with eubacteria [2,4].
The specific common ancestry of archaebacteria
and eukaryotes implied by quantitative
analyses of
macromolecular
sequences [2,4] extends to gene content. ‘Eukaryotic’ gene products encoded by archaebacterial
(but not eubacterial)
genomes include
TFIIB- and TBP-like transcription factors [5,6], subMicrobiological
Societies. Published
by Elsevier Science B.V
152
M.A.
Ragan
rl al. I FEMS
Microbiology
units of RNA polymerase [7], translation initiation
factors [8], ribosomal proteins [9], and a VCP-like
two-domain ATPase that in eukaryotes is involved
in cell-cycle regulation [lo]. Thus, an appropriate
archaebacterial genome could be a better ‘prokaryotic model of the eukaryotic genome’ than could any
eubacterial genome.
Sulfolobus solfataricus has one of the larger genames known among archaea ([1 I] and unpublished), and some experimental genetics has been developed [12]. Because Sulfolobus species thrive in
sulfur hot springs at 70-87°C pH 24 [13], all proteins in these organisms must be resistant to denaturation at this temperature and, by extension, under
other extreme conditions. These organisms can also
oxidize elemental sulfur to sulfuric acid, metabolize
organosulfur
compounds,
oxidize mineral sulfides,
and carry out a variety of other biochemically interesting reactions, including several of potential industrial interest [13,14]. All of these factors make S.
solfataricus a promising candidate for complete genome characterization;
we have now sequenced
more 1 Mbp (more than one-third) of its genome
[9]. In the course of this work, we discovered an
open reading frame (pelA) homologous with several
eukaryotic sequences, two of which (the pelota gene
of Drosophila melanogaster [ 151 and the dom34 gene
of Saccharomyces cerevisiae [ 16,171) are involved in
meiotic cell division. Herein we describe pelA, the
first prokaryotic homolog of pelota and dom34.
2. Materials and methods
A genomic library was prepared by digesting
DNA from S. solfataricus DSM 1617 with Hind111
to a mean size of 40-45 kb, and cloning into Escherichia coli using the cosmid vector Tropist3 [ 181. Cosmids were sorted by the restriction landmark strategy to yield a provisional
set of minimally
overlapping cosmid clones [9]. Two overlapping cosmids were selected, individually subcloned into pUC
18 by nebulization
and blunt-end ligation [9], and
sequenced using automated fluorescent techniques.
Contigs were linked, ambiguities resolved, and database searches conducted as detailed elsewhere [9].
Predicted proteins were aligned using ClustalW [19]
followed by modest visual adjustment.
Letters
144 (1996)
151-155
3. Results and discussion
Nucleotide sequences of the two cosmid inserts
were assembled into a single 56 105base pair contig
containing 71 open reading frames (ORFs) at least
300 base pairs in length [9]. Approximately
38 of
these ORFs could be identified, either solidly on
the basis of strong matches with well-identified database entries, or provisionally based on matches with
less well-identified entries, weaker matches, and/or
the presence of characteristic motifs. One of the provisionally identified ORFs, ORF ~04039, is located
between positions 33 827 and 32 793 on the complementary strand of this 56-kb contig [9].
Blastx analysis [20] of ORF co4039 against the
molecular-sequence
databases returned match probabilities of about 9 X lOe-16 with pelota protein
from
D.
melanogaster
[15], 4 X lOe- 13 with
DOM34 protein from S. cerevisiae [ 16,171, and
1 X lOe-13
with the hypothetical
protein R74.6
from Caenorhabditis elegans [21], levels significantly
greater than expected by chance for proteins of this
length. No matches were recorded against eubacterial entries, although the eubacterial database includes
the complete genomes of Haemophilus influenzae and
Mycoplasma genitalium. We conclude that pelota
homologs are either absent from eubacteria, or are
so highly changed as to be unrecognizable.
Within the multiple alignment (Fig. l), pairwise
comparison
of the S. solfataricus co4039 protein
(PelA) with pelota shows 76 identities (22.2%) and
73 conservative changes (21.3%) over 343 alignable
positions, with eight gaps. Comparing PelA with the
three full-length eukaryotic proteins (pelota, DOM34
and R74.6), 33 positions (9.6%) are identical in all
four, and a further 38 (11.1%) are identical in PelA
and two of the three others. These identical residues
are distributed fairly uniformly along the length of
the proteins, giving further support for homology of
pelA and pelota. By comparison,
89 of 378 positions
(23.5%) are identical among the three complete eukaryotic sequences, with no gaps.
In addition to the three complete eukaryotic pelota homologs, several other related sequences were
identified. Searches of the Expressed Sequence Tag
(EST) division of GenBank revealed five sequences
from Homo sapiens, one from Rattus sp. and one
from Arabidopsis thaliana. Four of the five Homo
M.A. Ragan et al. IFEMS Microbiology Letters 144 (1996) 151-155
153
344
Wh,ta F..~.LQ.A.SQ.I..L.ISDN.FRCQWSL.I.S.~...KI~~~.~....A.L...R~
Cae"~lhb&s
R74.6 F..E1W.NR.EIPEL.T..L..~.FRI\QDI.T.RXYV.L".~.I(.~I~S~~.~...CPI.....~~
sacchuomyc*r Ix*134 W..EltE.VI(.A.Y..ISYL.~.H..NLI\P_ ~Sv.SNG.K..CQ....AC..RIPL~
EST
...K..?K.N-AM.~.L.ISDE.FRWCIWAT.SRW.LVDSVKEN ?.T.RI?SSLWSDEQ?SP..."A.....PVPELSDPEGDSSSE~
Homo
+ + ++ * ++r+X+IY*#*+
+
w *+X4+*
+ x
* + +X+
.x
Droropikih
.*
3%
381
386
. *
Fig. 1. Amino acid alignment of pelota homologs: Sulfolobus solfataricus PelA, GenBank accession number U67942; Drosophila melanogarter pelota [15], accession number U27197; Caenorhabditis elegans R74.6 [21], accession number 236238; Saccharomyces cerevisiae
M)M34 [16,17], accession number X771 14; Ratrus sp.-Homo sapiens EST (fused accession numbers H31427, T30453, T19552 and
T19553); Arabidopsis thaliana EST (accession number T20628); and Homo sapiens EST (accession number D20778). Gaps introduced for
alignment are indicated by dashes (-). A dot c) indicates identity with the corresponding position in PelA (top row). An asterisk (*) under
a column indicates that the same amino acid occurs in PelA and all three full-length eukaryotic sequences (pelota, R74.6 and DOM34); a
pound sign () indicates identity of PelA and two of the three eukaryotic sequences. A plus (+) indicates positions identical in the three
full-length eukaryotic sequences but not PelA. The putative nuclear localization signal (NLS) is indicated at pelota positions 168-172.
Question marks (?) in EST sequences are shown where the nucleotide sequences ambiguously predict the protein sequence; residues at the
termini of ESTs of dubious alignment were omitted. Cumulative amino acid positions are given to the right of the complete sequences.
ESTs and the Rattus EST sequences were significantly overlapping and, in these regions, were nearly
identical in nucleotide sequence (data not shown).
We assembled these five sequences into a single representative sequence shown in Fig. 1 as ‘RattusHomo’. The remaining
Homo sequence could not
be assembled with Rattus-Homo; it may represent a
distinct gene, and is presented separately in Fig. 1.
One additional
S. cerevisiae homolog, XYZ3, was
revealed by tBlastn [20] searches of GenBank; it contains numerous single-nucleotide
insertions and deletions, and may be a pseudogene copy of the dom34
locus (cited in [16]). We PCR-amplified,
cloned and
sequenced XYZ3 from S. cerevisiae (data not shown)
and confirmed it to be a probable psuedogene [16].
S. solfataricus PelA is predicted to be a 39.6-kDa
protein with pZ 5.10. Structural predictions using the
methods of Gamier [22] and GGBSM [23] reveal it
to be a rather nondescript,
mostly helical protein
interrupted by four or more regions of coil. In one
turn- or coil-rich region of PelA (Fig. 1, pelota positions 168-172), a canonical eukaryotic nuclear localization signal PRKRK [ 151 is conserved precisely in
the Drosophila, Caenorhabditis and Homo sequences,
and is at least partially present in the Arubidopsis
protein, but is absent from the predicted PelA protein. The negatively charged carboxy-terminus
characteristic of all these eukaryotic pelota homologs is
likewise absent from PelA. Both of these features are
thus predicted to reflect the presence or function of
this protein
specifically within eukaryotic
cells.
Although there are no extensive regions of identity
between PelA and the eukaryotic proteins, one region of apparent conservation appears at the extreme
carboxy end of PelA, just prior to the negatively
charged terminus of the eukaryotic proteins; this region (in pelota, positions 374-383), which is even
more conserved among the eukaryotic
sequences,
could reflect a conserved ancestral function. These
and other residues that are conserved between PeiA
154
M.A. Rugan et al. I FEMS
Microbiology
and its eukaryotic homologs are potential targets for
site-directed mutagenesis and functional characterization.
Pelotu was first identified as a male-sterile mutant
in D. melanoguster, and subsequent work has demonstrated its involvement
in meiotic cell division
[15]. Strong mutant alleles of pelota result in meiotic
arrest of spermatocytes, although mitotic division in
spermatogenesis
appears normal [15]. On the other
hand, dom34 mutants of S. cerevisiae suggest both
mitotic and meiotic functions for the DOM34 protein, since they result in slowing of the mitotic cell
cycle, acceleration of meiotic divisions, and reduction of spore production (cited in [15]). Introduction
of the D. melanogaster pelota gene into S. cerevisiae
substantially rescues growth defects arising from mutations in dom34 (cited in [15]), incidentally providing further evidence that pelota and dom34 are
homologs. It might be more difficult to examine
whether S. solfataricus pelA can likewise rescue
dom34 or pelota mutants, as the putative nuclear
localization signal (and perhaps other necessary features of the protein, such as the negatively charged
carboxy-terminal
extension) are absent, and because
the PelA protein is presumably adapted to function
at elevated temperature;
functional characterization
might better be approached by mutagenesis experiments in cultures of S. solfataricus.
The discovery of a pelota homolog in an archaebacterium provides evidence that some of the machinery involved in meiosis predates the origin of
meiosis itself, which presumably arose after the divergence of eukaryotes from archaebacteria.
Thus,
the meiosis-specific role(s) of some eukaryotic proteins, including pelota, most likely arose within the
eukaryotic lineage, being recruited from some other
function(s).
Examination
of the function of such
meiotic-protein
homologs in prokaryotes may thus
allow a better understanding
not only of the role
of their eukaryotic counterparts in meiosis, but perhaps also of the evolutionary origin of meiosis itself.
To our knowledge, the only other identified archaebacterial homologs of meiotic genes are the radA
genes, themselves homologs of eubacterial recA [24]
and the meiosis-specific DMCI gene of S. cerevisiae
[25]. By contrast, no pelA homolog could be identified in eubacteria, suggesting that this gene, like cer-
Letters 144 (1996)
151-155
tain others [5-lo], may be specific to the archaebacterial-eukaryotic
lineage.
4. Note added in proof
After this paper was accepted, the complete genome sequence of the euryarchaeote Methanococcus
jannaschii was reported (Bult et al. (1996) Science
273, 105%1073) revealing a pelota homolog which
is 34% identical to S. solfataricus reported here.
Acknowledgments
We gratefully acknowledge the expert assistance of
G. Allard, C.C.-Y. Chan, T. Gaasterland, Q.Y. Liu,
S.L. Penny, M.E. Schenk, R.K. Singh and F. Young,
and critical review by S.L. Baldauf and M.E. Reith.
J. Chew and M. Dobson kindly provided S. cerevisiae DNA. This project was supported by the Canadian Genome Analysis and Technology Program, the
Canadian Institute for Advanced Research, the National Research Council of Canada, and the Medical
Research Council of Canada. J.M.L. is supported by
a Postdoctoral
Research Fellowship in Molecular
Evolution from the Alfred P. Sloan Foundation.
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